Distinct ultrastructural phenotypes of glial and neuronal alpha-synuclein inclusions in multiple system atrophy.

Multiple System Atrophy is characterized pathologically by the accumulation of alpha-synuclein (aSyn) into glial cytoplasmic inclusions (GCIs). The mechanism underlying the formation of GCIs is not well understood. In this study, correlative light and electron microscopy was employed to investigate aSyn pathology in the substantia nigra and putamen of post-mortem multiple system atrophy brain donors. Three distinct types of aSyn immuno-positive inclusions were identified in oligodendrocytes, neurons and dark cells presumed to be dark microglia. Oligodendrocytes contained fibrillar GCIs that were consistently enriched with lysosomes and peroxisomes, supporting the involvement of the autophagy pathway in aSyn aggregation in multiple system atrophy. Neuronal cytoplasmic inclusions exhibited ultrastructural heterogeneity resembling both fibrillar and membranous inclusions, linking multiple systems atrophy and Parkinson's disease. The novel aSyn pathology identified in the dark cells, displayed GCI-like fibrils or non-GCI-like ultrastructures suggesting various stages of aSyn accumulation in these cells. The observation of GCI-like fibrils within dark cells suggests these cells may be an important contributor to the origin or spread of pathological aSyn in multiple system atrophy. Our results suggest a complex interplay between multiple cell types that may underlie the formation of aSyn pathology in multiple system atrophy brain and highlight the need for further investigation into cell-specific disease pathologies in multiple system atrophy.

Parvalbumin interneurons in human ventromedial prefrontal cortex: a comprehensive post-mortem study of myelination and perineuronal nets in neurotypical individuals and depressed suicides with and without a history of child abuse.

Cortical parvalbumin interneurons (PV+) are major regulators of excitatory/inhibitory information processing, and their maturation is associated with the opening of developmental critical periods (CP). Recent studies reveal that cortical PV+ axons are myelinated, and that myelination along with perineuronal net (PNN) maturation around PV+ cells is associated with the closures of CP. Although PV+ interneurons are susceptible to early-life stress, their relationship between their myelination and PNN coverage remains unexplored. This study compared the fine features of PV+ interneurons in well-characterized human post-mortem ventromedial prefrontal cortex samples (n = 31) from depressed suicides with or without a history of child abuse (CA) and matched controls. In healthy controls, 81% of all sampled PV+ interneurons displayed a myelinated axon, while a subset (66%) of these cells also displayed a PNN, proposing a relationship between both attributes. Intriguingly, a 3-fold increase in the proportion of unmyelinated PV+ interneurons with a PNN was observed in CA victims, along with greater PV-immunofluorescence intensity in myelinated PV+ cells with a PNN. This study, which is the first to provide normative data on myelination and PNNs around PV+ interneurons in human neocortex, sheds further light on the cellular and molecular consequences of early-life adversity on cortical PV+ interneurons.

From flesh to bones: Multi-omics approaches in forensic science.

Recent advancements in omics techniques have revolutionised the study of biological systems, enabling the generation of high-throughput biomolecular data. These innovations have found diverse applications, ranging from personalised medicine to forensic sciences. While the investigation of multiple aspects of cells, tissues or entire organisms through the integration of various omics approaches (such as genomics, epigenomics, metagenomics, transcriptomics, proteomics and metabolomics) has already been established in fields like biomedicine and cancer biology, its full potential in forensic sciences remains only partially explored. In this review, we have presented a comprehensive overview of state-of-the-art analytical platforms employed in omics research, with specific emphasis on their application in the forensic field for the identification of the cadaver and the cause of death. Moreover, we have conducted a critical analysis of the computational integration of omics approaches, and highlighted the latest advancements in employing multi-omics techniques for forensic investigations.

Stability of Ni-Fe-Layered Double Hydroxide Under Long-Term Operation in AEM Water Electrolysis.

Anion exchange membrane water electrolysis (AEMWE) is an attractive method for green hydrogen production. It allows the use of non-platinum group metal catalysts and can achieve performance comparable to proton exchange membrane water electrolyzers due to recent technological advances. While current systems already show high performances with available materials, research gaps remain in understanding electrode durability and degradation behavior. In this study, the performance and degradation tracking of a Ni3Fe-LDH-based single-cell is implemented and investigated through the correlation of electrochemical data using chemical and physical characterization methods. A performance stability of 1000 h, with a degradation rate of 84 µV h-1 at 1 A cm-2 is achieved, presenting the Ni3Fe-LDH-based cell as a stable and cost-attractive AEMWE system. The results show that the conductivity of the formed Ni-Fe-phase is one key to obtaining high electrolyzer performance and that, despite Fe leaching, change in anion-conducting binder compound, and morphological changes inside the catalyst bulk, the Ni3Fe-LDH-based single-cells demonstrate high performance and durability. The work reveals the importance of longer stability tests and presents a holistic approach of electrochemical tracking and post-mortem analysis that offers a guideline for investigating electrode degradation behavior over extended measurement periods.

Reliable detection of RNA in hippocampus sections of mice by FISH up to a post-mortem delay of 24 h.

Proteins can be successfully localized in post-mortem (PM) brain tissue sections if the time until PM tissue sampling is not too long. In this study, we show that this also applies to the localization of RNA and in particular to the RNA of microglia-specific receptor proteins using the probes and the RNAscope™ Multiplex Fluorescent Detection Kit v2 from Advanced Cell Diagnostics. Brains were removed from killed mice after different PM delays and processed into paraffin sections. In sections of brains from animals whose cadavers had been kept at room temperature (21 °C) before tissue removal, ubiquitously expressed RNAs of genes with low to high expression levels (Polr2a, PPIB, and UBC) were reliably detected in the brain sections even if tissue removal was delayed by up to 48 h. In addition, microglia-specific G protein-coupled receptor RNA (Gpr34, P2ry12) could be reliably assigned to microglia by simultaneous labeling of the microglia with microglia-specific antibodies (Iba1 or P2ry12). Only after a delay of 48 h until tissue removal were the receptor RNA signals significantly lower. The reduction in receptor RNA signals could be delayed if the animal cadavers were stored at 4 °C until the brains were removed. Tissue sections of PM brain samples allow the spatial and cellular localization of specific RNA, at least if the sampling takes place within the first 24 h of PM.

Measles vaccination - An underestimated prevention measure: Analyzing a fatal case in Hildesheim, Germany.

Measles and rubella are targeted for elimination in the WHO region Europe. To reach the elimination goal, vaccination coverage of 95% must be achieved and sustained, the genotype information has to be provided for 80% of all outbreaks and transmission chains of a certain variant must not be detected for >12 months. The latter information is collected at Germany's National Reference Center Measles, Mumps, Rubella (NRC MMR). We describe here an outbreak of measles occurring in Hildesheim. The outbreak comprised 43 cases and lasted 14 weeks. Surprisingly, a high number of vaccination failures was observed since 11 cases had received two doses of the MMR vaccine and 4 additional cases were vaccinated once. A 33-year-old woman passed away during the outbreak. She was the mother of 5 children between 4 and 16 years of age. Two schoolchildren contracted measles and passed it on to the rest of the family. Due to delivery bottlenecks, the vaccination of the mother was delayed. She developed measles-like symptoms 3 days after vaccination and was found dead on the morning of day 8 after vaccination. A post-mortem examination was done to identify the cause of death. Moreover, molecular characterization of the virus was performed to analyze whether she was infected by the wildtype virus circulating at that time in Hildesheim or whether the vaccine may have been a concomitant and aggravating feature of her death. The result showed that the samples taken from her at the time of death and during necropsy contained the wildtype measles virus variant corresponding to MVs/Gir Somnath.IND/42.16 (WHO Seq-ID D8-4683) that fueled the Hildesheim outbreak and circulated in Germany from March 2018 to March 2020. The vaccine virus was not detected. Moreover, two aspects uncovered by the post-mortem examination were remarkable; the woman died from giant cell pneumonia, which is a complication seen in immune-suppressed individuals and she was actively using cannabis. THC is known to influence the immune system, but literature reports describing the effects are limited.

Changes in the locus coeruleus during the course of Alzheimer's disease and their relationship to cortical pathology.

AIMS: In Alzheimer's disease (AD), the locus coeruleus (LC) undergoes early and extensive neuronal loss, preceded by abnormal intracellular tau aggregation, decades before the onset of clinical disease. Neuromelanin-sensitive MRI has been proposed as a method to image these changes during life. Surprisingly, human post-mortem studies have not examined how changes in LC during the course of the disease relate to cerebral pathology following the loss of the LC projection to the cortex. METHODS: Immunohistochemistry was used to examine markers for 4G8 (pan-Aß) and AT8 (ptau), LC integrity (neuromelanin, dopamine ß-hydroxylase [DßH], tyrosine hydroxylase [TH]) and microglia (Iba1, CD68, HLA-DR) in the LC and related temporal lobe pathology of 59 post-mortem brains grouped by disease severity determined by Braak stage (0-II, III-IV and V-VI). The inflammatory environment was assessed using multiplex assays. RESULTS: Changes in the LC with increasing Braak stage included increased neuronal loss (p < 0.001) and microglial Iba1 (p = 0.005) together with a reduction in neuromelanin (p < 0.001), DßH (p = 0.002) and TH (p = 0.041). Interestingly in LC, increased ptau and loss of neuromelanin were detected from Braak stage III-IV (p = 0.001). At Braak stage V/VI, the inflammatory environment was different in the LC vs TL, highlighting the anatomical heterogeneity of the inflammatory response. CONCLUSIONS: Here, we report the first quantification of neuromelanin during the course of AD and its relationship to AD pathology and neuroinflammation in the TL. Our findings of neuromelanin loss early in AD and before the neuroinflammatory reaction support the use of neuromelanin-MRI as a sensitive technique to identify early changes in AD.

Congenital heart diseases (CHDs) and forensic investigations: Searching for the cause of death.

Congenital Heart Diseases (CHDs) are a group of structural abnormalities or defects of the heart that are present at birth. CHDs could be connected to sudden death (SD), defined by the WHO (World Health Organization) as "death occurring within 24 h after the onset of the symptoms" in an apparently "healthy" subject. These conditions can range from relatively mild defects to severe, life-threatening anomalies. The prevalence of CHDs varies across populations, but they affect millions of individuals worldwide. This article aims to discuss the post-mortem investigation of death related to CHDs, exploring the forensic approach, current methodologies, challenges, and potential advancements in this challenging field. A further goal of this article is to provide a guide for understanding these complex diseases, highlighting the pivotal role of autopsy, histopathology, and genetic investigations in defining the cause of death, and providing evidence about the translational use of autopsy reports. Forensic investigations play a crucial role in understanding the complexities of CHDs and determining the cause of death accurately. Through collaboration between medical professionals and forensic experts, meticulous examinations, and analysis of evidence, valuable insights can be gained. These insights not only provide closure to the families affected but also contribute to the prevention of future tragedies.

Forensic age estimation of the knee by post-mortem DR, CT, and MR imaging: a comparative study.

It is believed by many that reference data for age estimation purposes must be imaging-modality specific. A study from our department has however proven otherwise. We therefore found it interesting to investigate this further by looking at the level of agreement between different imaging modalities. The aim of this study was to investigate the level of agreement between the three radiological modalities, computed tomography (CT), magnetic resonance imaging (MRI), and digital radiography (DR), in assessing the ossification of the epiphyses of the knee. A total of 34 deceased individuals of 10-25 years of age, brought in for a medicolegal autopsy at our department, were scanned by CT, MRI, and DR. The ossification stages of the three bones of the right knee, distal femoral, proximal tibial, and proximal fibular epiphysis were assessed using the established combined staging method by Schmeling et al. and Kellinghaus et al. Analysis of the results by Cohen's weighted kappa showed a good agreement between CT and DR (K = 0.61-0.70), and MRI and DR (K = 0.68-0.79) but only moderate agreement between CT and MRI (K = 0.55-0.57). This leads us to conclude that different radiological images cannot be used interchangeably for age estimation purposes, so reference material needs to be imaging-modality specific. However, to make a more general conclusion research on a larger population is needed.

Parasitic and predatory behavior of Alysia manducator (Hymenoptera: Braconidae) on blow fly larvae feeding on an adult pig carcass in the Western Cape Province of South Africa: preliminary observations and forensic implications.

Wasps are part of the entomofauna associated with vertebrate carrion. They are known to parasitize and prey on specific life stages of insect hosts such as eggs, larvae, pupae, and/or adults associated with vertebrate carrion. However, reports of parasitic behavior of wasps on carrion-associated insect life stages and their possible forensic implications are non-existent in the Western Cape Province of South Africa. This study is part of ongoing research investigating the entomofauna and their pattern of succession on an adult pig carcass in Cape Town, South Africa. During this study, the parasitic wasp Alysia manducator was noted parasitizing and preying on blow fly larvae associated with the decomposing carcass. The arrival of A. manducator coincides with the occurrence of blow fly eggs and/or larvae on the carcass. These wasps were seen in close association with the eggs and larvae of blow flies on various parts of the carcass and some wasps were seen dragging fly larvae attached to their ovipositors away from one part of the carcass to another. Some A. manducator were also observed walking over several larvae on the carcass while exhibiting a stabbing behavior presumably in search of a host for oviposition. We suggest that the observations recorded in this study are of considerable forensic importance as the dragging effect and predatory and stabbing behavior exhibited by A. manducator could potentially disrupt the feeding and development of the fly larvae on the carcass. This could subsequently alter the process of carcass decomposition and/or affect minimum post-mortem interval estimations.

Epigenetic analyses in forensic medicine: future and challenges.

The possibility of using epigenetics in forensic investigation has gradually risen over the last few years. Epigenetic changes with their dynamic nature can either be inherited or accumulated throughout a lifetime and be reversible, prompting investigation of their use across various fields. In forensic sciences, multiple applications have been proposed, such as the discrimination of monozygotic twins, identifying the source of a biological trace left at a crime scene, age prediction, determination of body fluids and tissues, human behavior association, wound healing progression, and determination of the post-mortem interval (PMI). Despite all these applications, not all the studies considered the impact of PMI and post-sampling effects on the epigenetic modifications and the tissue-specificity of the epigenetic marks.This review aims to highlight the substantial forensic significance that epigenetics could support in various forensic investigations. First, basic concepts in epigenetics, describing the main epigenetic modifications and their functions, in particular, DNA methylation, histone modifications, and non-coding RNA, with a particular focus on forensic applications, were covered. For each epigenetic marker, post-mortem stability and tissue-specificity, factors that should be carefully considered in the study of epigenetic biomarkers in the forensic context, have been discussed. The advantages and limitations of using post-mortem tissues have been also addressed, proposing directions for these innovative strategies to analyze forensic specimens.

An effective method for preparing histological sections of blow fly intra-puparial stages for minimum PMI estimations.

Blow flies (Diptera: Calliphoridae) are generally early colonisers of fresh cadavers, enabling the estimation of a minimum post-mortem interval (minPMI) based on an accurate aging of the oldest immature stages associated with a cadaver. In blow flies, the pupal stage and the subsequent development of the adult take place inside a protective case, the puparium, formed from the hardened and darkened cuticle of the third instar larva. Because the puparium is an opaque structure that shows virtually no external changes, qualitative analyses of the internal tissues can be very informative for determining reliable age-specific morphological markers. Those analyses can be performed using either non-invasive but expensive and not widely accessible techniques, or traditional histological methods, which are invasive as they require the serial sectioning of the sample. Histological methods are often readily available for forensic researchers and practitioners; however, the histological study of blow fly intra-puparial stages has traditionally been hampered by the poor paraffin infiltration of tissues due to the abundance of fat bodies, resulting in usually fragmented sections and the subsequent loss of relevant information. We present here an effective method for the preparation of histological sections of blow fly intra-puparial stages, maximising the paraffin infiltration while enabling the production of clean and entire sections that allow for the use of reliable age-specific morphological markers, thus improving the accuracy of minPMI estimations when access to more costly techniques is not feasible.

Prediction model for anabolic androgenic steroid positivity in forensic autopsy cases - a new tool to the autopsy room.

Non-prescription use of anabolic androgenic steroids (AAS) is associated with an increased risk of premature death. However, these substances are seldom screened in connection with forensic cause-of-death investigation, unless the forensic pathologist specifically suspects use, often based on a positive AAS use history. Since AAS use is often concealed from others, this practice may lead to mistargeting of these analyses and significant underestimation of the true number of AAS positive cases undergoing forensic autopsy. Thus, more accurate diagnostic tools are needed to identify these cases. The main objective of this study was to determine, whether a multivariable model could predict AAS urine assay positivity in forensic autopsies. We analyzed retrospectively the autopsy reports of all cases that had been screened for AAS during forensic cause-of-death investigation between 2016-2019 at the Finnish Institute for Health and Welfare forensic units (n = 46). Binary logistic regression with penalized maximum likelihood estimation was used to generate a nine-variable model combining circumferential and macroscopic autopsy-derived variables. The multivariable model predicted AAS assay positivity significantly better than a "conventional" model with anamnestic information about AAS use only (area under the receiver operating characteristic curve [AUC] = 0.968 vs. 0.802, p = 0.005). Temporal validation was conducted in an independent sample of AAS screened cases between 2020-2022 (n = 31), where the superiority of the multivariable model was replicated (AUC = 0.856 vs. 0.644, p = 0.004). Based on the model, a calculator predicting AAS assay positivity is released as a decision-aiding tool for forensic pathologists working in the autopsy room.

Fatal child abuse detected by systematic post-mortem fundus photograph in sudden death in infancy.

In living children, the use of a wide field fundus camera such as RetCam is the gold standard practice to document retinal haemorrhages in suspected cases of abusive head trauma (AHT). In case of sudden unexpected death in infancy (SUDI), child abuse must be considered as a possible cause of death and an eye examination is required. However, no example of post-mortem fundus photograph (PMFP) of retinal haemorrhages related to AHT is yet available for clinicians.We report a SUDI case, with no external traumatic lesions or limb fractures, for which prompt PMFP showed retinal haemorrhages typical of AHT: child abuse was subsequently confirmed by the forensic investigation. We discuss why PMFP is a relevant screening test to detect retinal haemorrhages in the case of SUDI and why the use of the RetCam should be further investigated.

The influence of cooling on biomechanical time since death estimations using ovine brain tissue.

The significance of biomechanical analyses for forensic time since death estimations has recently been demonstrated. Previous biomechanical analyses successfully discriminated post-mortem brain tissue from tissue with a post-mortem interval of at least one day when held at 20 °C. However, the practical utility of such analyses beyond day one at 20 °C was limited. This study investigates the storage, loss, and complex shear modulus of various brain regions in sheep stored at 4 °C in 24-hour intervals over four days post-mortem using rheometry tests. The aim is to identify the critical biomechanical tissue property values to predict post-mortem time and assess the temperature sensitivity of the rheometry method by comparing results to recent findings at 20 °C. Thirty sheep brains were examined, including the frontal lobe, parietal lobe, anterior and posterior deep brain, superior colliculi, pons, medulla, and cerebellum. Rheometry tests were conducted, and receiver operator characteristic analyses were employed to establish cut-off values. At 4 °C storage, all investigated biomechanical properties of the examined brain regions remained stable for at least one day post-mortem. Using cerebellar samples stored at 4 °C, a post-mortem interval of at least two days could be determined with excellent diagnostic ability. Complex shear modulus values below 1435 Pa or storage modulus values below 1313 Pa allowed prediction of two or more days post-mortem. Comparisons between 4 °C and 20 °C revealed brain region-specific results. For instance, the complex shear moduli of the anterior deep brain at 4 °C were significantly higher on all individual testing days when compared to 20 °C. In contrast, the combined medulla and pons samples were similar on each day. Rheometry testing of brain tissue consistently stored at 4 °C since death proved valuable for forensic time since death estimations starting from two days after death.

Investigating cardiac genetic background in sudden infant death syndrome (SIDS).

Sudden infant death syndrome (SIDS) is still the leading cause of death for newborns in developed countries. The pathophysiological mechanisms have not been fully clarified, but in some of SIDS cases variants of genes associated with inherited cardiac conditions are found. In this study, an analysis of SCD-related genes was performed to determine the prevalence of rare pathogenic (P) or likely pathogenic (LP) variants that could provide an unambiguous explanation for the fatal event. A cohort of 76 SIDS cases underwent Next-Generation Sequencing (NGS) analysis with a custom panel of SCD-related genes. Rare variants were classified according to the guidelines provided by the American College of Medical Genetics and Genomics (ACMG) and the specifications of the ClinGen association. Post-mortem genetic testing identified 50 (65.8%) carriers of at least one variant in SCD genes. 104 rare genetic variants were found, 65.4% in genes encoding structural proteins. Only 4 out of 76 cases (5.3%) hosted at least a P or LP variant found in genes with structural or structural/arrhythmogenic functions (SLC22A5, SCN5A, MYL3and TTN). 99 variants were classified as of uncertain significance (VUS). The difference in the distribution of variants between gene groups by function was not statistically significant (chi square, p = 0,219). Despite this, most of the variants concerned structural genes that were supposed to have a close interaction with ion channels, thus providing an explanation for the arrhythmic event. Segregation analysis, reclassification of VUS variants and identification of new associated genes could clarify the implications of the current findings.

Catch me if you can-emission patterns of human bodies in relation to postmortem changes.

The present study examines for the first time the emission patterns and olfactory signatures of 9 complete human corpses of different stages of decomposition. Air sampling was performed inside the body bags with solid sorbents and analysed by coupled gas chromatography-mass spectrometry after thermal desorption (TD-GC-MS). Furthermore, odour-related substances were detected by gas chromatography-olfactometry (GC-O). Sulfurous compounds (mainly dimethyl di- and trisulfide) were identified as most important to the odour perception. Around 350 individual organic substances were detected by TD-GC-MS, notably sulfurous and nitrogenous substances as well as branched alkanes, aldehydes, ketones, alcohols, carboxylic acids, carboxylic acid esters and ethers. A range of terpenes was detected for the first time in a characteristic emission pattern over all decomposition stages. Concentrations of the substances varied greatly, and no correlation between the emission patterns, the stage of decomposition and the cause of death could be found. While previous studies often analysed pig cadavers or only parts of human tissue, the present study shows the importance of analysing complete human corpses over a range of decomposition stages. Moreover, it is shown that using body bags as a kind of "emission test chamber" is a very promising approach, also because it is a realistic application considering the usual transport and store of a body before autopsy.

Concordance between MITS and conventional autopsies for pathological and virological diagnoses.

In pandemics or to further study highly contagious infectious diseases, new strategies are needed for the collection of post-mortem tissue samples to identify the pathogen as well as its morphological impact. In this study, an ultrasound-guided minimally invasive tissue sampling (MITS) protocol was developed and validated for post-mortem use. The histological and microbiological qualities of post-mortem specimens were evaluated and compared between MITS and conventional autopsy (CA) in a series of COVID-19 deaths. Thirty-six ultrasound-guided MITS were performed. In five cases more, specimens for histological and virological examination were also obtained and compared during the subsequently performed CA. Summary statistics and qualitative interpretations (positive, negative) were calculated for each organ tissue sample from MITS and CA, and target genes were determined for both human cell count (beta-globin) and virus (SARS-CoV-2 specific E gene). There are no significant differences between MITS and CA with respect to the detectability of viral load in individual organs, which is why MITS can be of utmost importance and an useful alternative, especially during outbreaks of infectious diseases.

Forensic taphonomic experimental design matters: a study assessing clothing and carrion biomass load on scavenging in Cape Town, South Africa.

The identification of unknown human remains is a significant and ongoing challenge in South Africa, worsened by the country's high murder rate. The rate of decomposition in South Africa is significantly influenced by vertebrate scavenging, which, if not considered, can impede the accurate estimation of the post-mortem interval. Scavenging patterns vary greatly depending on the environment and ecological region, and there is limited data for the Western Cape province. To address this gap, two clothed and uncaged pig carcasses weighing 60 kg each were placed in the field in July 2021 and January 2022, respectively. Motion-activated infrared-capable trail cameras were used to observe decomposition, scavenger species, and their activities. Additionally, a comparative sample of 16 unclothed carcasses deployed between 2014 and 2016 in the same habitat were analyzed to assess the impact of clothing and biomass load. The study found three main results: (1) Regardless of habitat or biomass load, it took significantly less time to reach decomposition milestones (25%, 50%, and 75%) during the summer season; (2) the presence of mongoose scavengers had a greater impact on the time required to reach milestones during winter compared to summer; and (3) single carcass deployments reached the milestones faster than multi-carcass deployments in both seasons. This research highlights the potential inaccuracy of current methods for estimating the post-mortem interval when scavenging activity is not considered or documented in the underlying experimental data, particularly for environments or ecological biomes where scavengers actively impact decomposition rates.

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.