The biodistribution of triamcinolone acetonide injections in severe keloids: an exploratory three-dimensional fluorescent cryomicrotome study.

Intralesional corticosteroid injections are a first-line treatment for keloids; yet clinical treatment results are highly variable and often suboptimal. Variation in triamcinolone acetonide (TAC) biodistribution may be an important reason for the variable effects of TAC treatment in keloids. In this exploratory study we investigated the biodistribution of TAC in keloids and normal skin using different drug delivery techniques. Fluorescent-labeled TAC suspension was administered into keloids and normal skin with a hypodermic needle and an electronic pneumatic jet injector. TAC biodistribution was represented by the fluorescent TAC volume and 3D biodistribution shape of TAC, using a 3D-Fluorescence-Imaging Cryomicrotome System. Twenty-one keloid and nine normal skin samples were analyzed. With needle injections, the mean fluorescent TAC volumes were 990 µl ± 479 in keloids and 872 µl ± 227 in normal skin. With the jet injector, the mean fluorescent TAC volumes were 401 µl ± 252 in keloids and 249 µl ± 67 in normal skin. 3D biodistribution shapes of TAC were highly variable in keloids and normal skin. In conclusion, TAC biodistribution in keloids is highly variable for both needle and jet injection. This may partly explain the variable treatment effects of intralesional TAC in keloids. Future research is needed to confirm this preliminary finding and to optimize drug delivery in keloids.

Subminute thermal damage to cell types present in the skin.

INTRODUCTION: Psoriasis is characterized by an increase in the proliferation of keratinocytes and nerve fiber activity, contributing to the typical skin lesions. Pulsed Dye Laser (PDL) treatment is effective for the treatment of psoriatic lesions but its mechanism remains unclear. One hypothesis is that PDL causes thermal damage by the diffusion of heat to neighboring structures in lesional skin. There is limited information on the thermal sensitivity of these neighboring skin cells when exposed to hyperthermia for durations lasting less than a minute. Our study aimed to investigate the cell-specific responses to heat using sub-minute exposure times and moderate to ablative hyperthermia. MATERIALS AND METHODS: Cultured human endothelial cells, smooth muscle cells, neuronal cells, and keratinocytes were exposed to various time (2-20 sec) and temperature (45-70 °C) combinations. Cell viability was assessed by measuring intracellular ATP content 24 h after thermal exposure and this data was used to calculate fit parameters for the Arrhenius model and CEM43 calculations. RESULTS: Our results show significant differences in cell survival between cell types (p < 0.0001). Especially within the range of 50-60 °C, survival of neuronal cells and keratinocytes was significantly less than that of endothelial and smooth muscle cells. No statistically significant difference was found in the lethal dose (LT50) of thermal energy between neuronal cells and keratinocytes. However, CEM43 calculations showed significant differences between all four cell types. CONCLUSION: The results imply that there is a cell-type-dependent sensitivity to thermal damage which suggests that neuronal cells and keratinocytes are particularly susceptible to diffusing heat from laser treatment. Damage to these cells may aid in modulating the neuro-inflammatory pathways in psoriasis. These data provide insight into the potential mechanisms of PDL therapy for psoriasis and advance our understanding of how thermal effects may play a role in its effectiveness.

Modeling pulsed dye laser treatment of psoriatic plaques by combining numerical methods and image-derived lesion morphologies.

OBJECTIVES: Knowledge of the physical effects of pulsed dye laser (PDL) treatment of psoriatic lesions is essential in unraveling the remedial mechanisms of this treatment and hence also in maximizing in its disease-modifying potential. Therefore, the main objective of this study was to provide estimates of these physical effects (for laser wavelengths of 585 and 595 nm), with the aim of identifying pathogenic processes that may be affected by these conditions. METHODS: We modeled the laser light propagation and subsequent photothermal heating by numerically solving the transient diffusion and heat equations simultaneously. To this end, we used the finite element method in conjunction with an image-derived psoriatic lesion morphology (which was defined by segmenting blood vessels from a confocal microscopy image of a fluorescently labeled section of a 3 mm punch biopsy of a psoriatic lesion). The resulting predictions of the generated temperature field within the lesion were then used to assess the possibility of stalling or arresting some suspected pathogenic processes. RESULTS: According to our results, it is conceivable that perivascular nerves are thermally denatured, as almost all locations that reach 60°C were found to be within 18 µm (at 585 nm) and 11 µm (at 595 nm) of a blood vessel wall. Furthermore, activation of TRPV1 and TRPV2 channels in perivascular neuronal and immune cells is highly likely, since a critical temperature of 43°C is generated at locations within up to 350 µm of a vessel wall (at both wavelengths) and sustained for up to 700 ms (at 585 nm) and 40 ms (at 595 nm), while a critical temperature of 52°C is reached by locations within 80 µm (at 585 nm) and 30 µm (at 595 nm) of a vessel wall and sustained for up to 100 ms (at 585 nm) and 30 ms (at 595 nm). Finally, we found that the blood vessel coagulation-inducing temperature of 70°C is sustained in the vascular epithelium for up to 19 and 5 ms at 585 and 595 nm, respectively, rendering partial or total loss of vascular functionality a distinct possibility. CONCLUSIONS: The presented approach constitutes a useful tool to provide realistic estimates of the photothermal effects of PDL treatment of psoriatic plaques (as well as other selective photothermolysis-based treatments), yielding information that is essential in guiding future experimental studies toward unraveling the remedial mechanisms of these treatments.

Home-Use Hyaluronic Acid Jet Injectors: Unreliable and Unsafe.

BACKGROUND: Needle-free hyaluronic acid (HA) jet injectors are gaining popularity for rejuvenation treatment. The devices are widely available online and are used for self-injection or in beauty salons by nonphysicians. However, little is known about their performance and safety. OBJECTIVE: To explore the injection efficiency and cutaneous biodistribution patterns administered with home-use compared with medical jet injectors and to assess safety aspects. MATERIALS AND METHODS: The authors injected HA into ex vivo human skin with 4 home-use and 2 medical injectors. The intracutaneous dose of HA was calculated, and the cutaneous biodistribution of HA was assessed using a 3-dimensional Fluorescent Imaging Cryomicrotome System (3D-FICS). Safety aspects were evaluated based on the presence of a manual, CE (conformité européenne) mark, and sterility. RESULTS: The intracutaneous dose delivered by the home-use injectors was markedly lower compared with the medical injectors. 3D imaging for home-use injectors showed superficial epidermal distribution with low distribution volumes. For medical injectors, volumes were substantially larger and mainly middermal. All evaluated safety aspects were lacking. CONCLUSION: Results of this study suggest that the specific combinations of home-use injectors and HA used in this study are unreliable and unsafe, which casts doubts on the performance of these treatments in general.

Analysis of the fluorescent properties of vaginal fluid upon ageing.

Detection and identification of body fluids are crucial aspects of forensic investigations, aiding in crime scene reconstructions and providing important leads. Although many methods have been developed for these purposes, no method is currently in use in the forensic field that allows rapid, non-contact detection and identification of vaginal fluids directly at the crime scene. The development of such technique is mainly challenged by the complex chemistry of the constituents, which can differ between donors and exhibits changes based on woman's menstrual cycle. The use of fluorescence spectroscopy has shown promise in this area for other biological fluids. Therefore, the aim of this study was to identify specific fluorescent signatures of vaginal fluid with fluorescence spectroscopy to allow on-site identification. Additionally, the fluorescent properties were monitored over time to gain insight in the temporal changes of the fluorescent spectra of vaginal fluid. The samples were excited at wavelengths ranging from 200 to 600 nm and the induced fluorescence emission was measured from 220 to 700 nm. Excitation and emission maps (EEMs) were constructed for eight donors at seven time points after donation. Four distinctive fluorescence peaks could be identified in the EEMs, indicating the presence of proteins, fluorescent oxidation products (FOX), and an unidentified component as the dominant contributors to the fluorescence. To further asses the fluorescence characteristics of vaginal fluid, the fluorescent signatures of protein and FOX were used to monitor protein and lipid oxidation reactions over time. The results of this study provide insights into the intrinsic fluorescent properties of vaginal fluid over time which could be used for the development of a detection and identification method for vaginal fluids. Furthermore, the observed changes in fluorescence signatures over time could be utilized to establish an accurate ageing model.

Towards Onsite Age Estimation of Semen Stains Using Fluorescence Spectroscopy.

The age estimation of biological traces is one of the holy grails in forensic investigations. We developed a method for the age estimation of semen stains using fluorescence spectroscopy in conjunction with a stoichiometric ageing model. The model describes the degradation and generation rate of proteins and fluorescent oxidation products (FOX) over time. The previously used fluorimeter is a large benchtop device and requires system optimization for forensic applications. In situ applications have the advantage that measurements can be performed directly at the crime scene, without additional sampling or storage steps. Therefore, a portable fiber-based fluorimeter was developed, consisting of two optimized light-emitting diodes (LEDs) and two spectrometers to allow the fluorescence protein and FOX measurements. The handheld fiber can be used without touching the traces, avoiding the destruction or contamination of the trace. In this study, we have measured the ageing kinetics of semen stains over time using both our portable fluorimeter and a laboratory benchtop fluorimeter and compared their accuracies for the age estimation of semen stains. Successful age estimation was possible up to 11 days, with a mean absolute error of 1.0 days and 0.9 days for the portable and the benchtop fluorimeters, respectively. These results demonstrate the potential of using the portable fluorimeter for in situ applications.

Temperature-specific spectral shift of luminescing thermally altered human remains.

Human bone has shown to have luminescent properties that remain throughout the phases of cremation, with the exception of fully carbonized bone, when excited with a narrow band light source. During this research, an alternate light source (420-470nm, peak at 445nm) was used to visualize and investigate latent details relevant for forensic investigations of human remains recovered at fire scenes. As fire is a destructive force, it induces a vast variety of physical and chemical alterations to all components of the bone, making the subsequent analysis and interpretation of burned human remains challenging. A spectral shift in emission bandwidth, from green to red, was previously observed when the exposure temperature increased from 700 to 800 °C. This spectral shift was reproduced on a total of 10 human forearms, divided into 20 segments, by burning at 700 °C and 900 °C in an ashing furnace. The shift of emission bandwidth caused only by an increase in temperature was furthermore investigated by colorimetric analysis, proving the spectral shift to be significant. By easily quantifying the spectral shift, substantiation is provided for the use of this technique in practice to improve the interpretation of heat induced changes of bone.

Specific fluorescent signatures for body fluid identification using fluorescence spectroscopy.

Non-invasive, rapid, on-site detection and identification of body fluids is highly desired in forensic investigations. The use of fluorescence-based methods for body fluid identification, have so far remain relatively unexplored. As such, the fluorescent properties of semen, serum, urine, saliva and fingermarks over time were investigated, by means of fluorescence spectroscopy, to identify specific fluorescent signatures for body fluid identification. The samples were excited at 81 different excitation wavelengths ranging from 200 to 600 nm and for each excitation wavelength the emission was recorded between 220 and 700 nm. Subsequently, the total emitted fluorescence intensities of specific fluorescent signatures in the UV-visible range were summed and principal component analysis was performed to cluster the body fluids. Three combinations of four principal components allowed specific clustering of the body fluids, except for fingermarks. Blind testing showed that 71.4% of the unknown samples could be correctly identified. This pilot study shows that the fluorescent behavior of ageing body fluids can be used as a new non-invasive tool for body fluid identification, which can improve the current guidelines for the detection of body fluids in forensic practice and provide the robustness of methods that rely on fluorescence.

Comparison of preprocessing techniques to reduce nontissue-related variations in hyperspectral reflectance imaging.

Significance: Hyperspectral reflectance imaging can be used in medicine to identify tissue types, such as tumor tissue. Tissue classification algorithms are developed based on, e.g., machine learning or principle component analysis. For the development of these algorithms, data are generally preprocessed to remove variability in data not related to the tissue itself since this will improve the performance of the classification algorithm. In hyperspectral imaging, the measured spectra are also influenced by reflections from the surface (glare) and height variations within and between tissue samples. Aim: To compare the ability of different preprocessing algorithms to decrease variations in spectra induced by glare and height differences while maintaining contrast based on differences in optical properties between tissue types. Approach: We compare eight preprocessing algorithms commonly used in medical hyperspectral imaging: standard normal variate, multiplicative scatter correction, min-max normalization, mean centering, area under the curve normalization, single wavelength normalization, first derivative, and second derivative. We investigate conservation of contrast stemming from differences in: blood volume fraction, presence of different absorbers, scatter amplitude, and scatter slope-while correcting for glare and height variations. We use a similarity metric, the overlap coefficient, to quantify contrast between spectra. We also investigate the algorithms for clinical datasets from the colon and breast. Conclusions: Preprocessing reduces the overlap due to glare and distance variations. In general, the algorithms standard normal variate, min-max, area under the curve, and single wavelength normalization are the most suitable to preprocess data used to develop a classification algorithm for tissue classification. The type of contrast between tissue types determines which of these four algorithms is most suitable.

Next-generation time of death estimation: combining surrogate model-based parameter optimization and numerical thermodynamics.

The postmortem interval (PMI), i.e. the time since death, plays a key role in forensic investigations, as it aids in the reconstruction of the timeline of events. Currently, the standard method for PMI estimation empirically correlates rectal temperatures and PMIs, frequently necessitating subjective correction factors. To address this shortcoming, numerical thermodynamic algorithms have recently been developed, providing rigorous methods to simulate postmortem body temperatures. Comparing these with measured body temperatures then allows non-subjective PMI determination. This approach, however, hinges on knowledge of two thermodynamic input parameters, which are often irretrievable in forensic practice: the ambient temperature prior to discovery of the body and the body temperature at the time of death (perimortem). Here, we overcome this critical limitation by combining numerical thermodynamic modelling with surrogate model-based parameter optimization. This hybrid computational framework predicts the two unknown parameters directly from the measured postmortem body temperatures. Moreover, by substantially reducing computation times (compared with conventional optimization algorithms), this powerful approach is uniquely suited for use directly at the crime scene. Crucially, we validated this method on deceased human bodies and achieved the lowest PMI estimation errors to date (0.18 h ± 0.77 h). Together, these aspects fundamentally expand the applicability of numerical thermodynamic PMI estimation.

Mechanical or thermal damage: differentiating between underlying mechanisms as a cause of bone fractures.

To investigate the differences between pre- and post-fire fractures, 30 human forearm bones were subjected to either blunt-force impact, burning, or both. Bones, covered in soft tissue and wrapped in clothing, were burned in a reconstructed house fire. The burning context and dynamics led to differential burning, that was equal amongst the three groups. To evaluate the damage to the bones, a data collection sheet was developed based on the current literature on fracture features. To analyze the relation between exposure temperature and fracture class and occurrence, color was measured to estimate the exposure temperature. Observable and measurable changes on bone, and fracture surfaces, were macro- and microscopically analyzed. Many features overlapped between the three groups, and thus are not usable for differentiation. Fractures caused by blunt force impact (post-mortem, pre-fire) showed a rough fracture surface with smoothness in curved/slanted regions near the margin after burning, while heat-induced bone fractures showed a smooth fracture surface. The margins and surface of bone fractures that originated after the fire (indirect heat-induced) were evenly discolored, whereas heat-induced bone fractures showed uneven discoloration of the fracture margin and surface. Although there were generally more heat-induced fractures in the 450-700 °C range, no other distinctive trend was observed between exposure temperature and class of fractures.

Separation of overlapping fingerprints by principal component analysis and multivariate curve resolution-alternating least squares analysis of hyperspectral imaging data.

Overlapping fingerprints are often found at crime scenes, but only individual fingerprints separated from each other are admissible as evidence in court. Fingerprint components differ slightly among individuals, and thus their fluorescence spectra also differ from each other. Therefore, the separation of overlapping fingerprints using the difference of the fluorescence spectrum was performed with a hyperspectral imager. Hyperspectral data (HSD) of overlapping fingerprints were recorded under UV LED excitation. Principal component analysis (PCA) and multivariate curve resolution-alternating least squares (MCR-ALS) were applied to the HSD to determine the optimal method for obtaining high-contrast images of individual fingerprints. The results suggested that MCR-ALS combined with PCA-based initialization is capable of separating overlapping fingerprints into individual fingerprints. In this study, a method for separating overlapping fingerprints without initial parameters was proposed.

Late Gadolinium Enhancement Cardiovascular Magnetic Resonance Assessment of Substrate for Ventricular Tachycardia With Hemodynamic Compromise.

Background: The majority of data regarding tissue substrate for post myocardial infarction (MI) VT has been collected during hemodynamically tolerated VT, which may be distinct from the substrate responsible for VT with hemodynamic compromise (VT-HC). This study aimed to characterize tissue at diastolic locations of VT-HC in a porcine model. Methods: Late Gadolinium Enhancement (LGE) cardiovascular magnetic resonance (CMR) imaging was performed in eight pigs with healed antero-septal infarcts. Seven pigs underwent electrophysiology study with venous arterial-extra corporeal membrane oxygenation (VA-ECMO) support. Tissue thickness, scar and heterogeneous tissue (HT) transmurality were calculated at the location of the diastolic electrograms of mapped VT-HC. Results: Diastolic locations had median scar transmurality of 33.1% and a median HT transmurality 7.6%. Diastolic activation was found within areas of non-transmural scar in 80.1% of cases. Tissue activated during the diastolic component of VT circuits was thinner than healthy tissue (median thickness: 5.5 mm vs. 8.2 mm healthy tissue, p < 0.0001) and closer to HT (median distance diastolic tissue: 2.8 mm vs. 11.4 mm healthy tissue, p < 0.0001). Non-scarred regions with diastolic activation were closer to steep gradients in thickness than non-scarred locations with normal EGMs (diastolic locations distance = 1.19 mm vs. 9.67 mm for non-diastolic locations, p < 0.0001). Sites activated late in diastole were closest to steep gradients in tissue thickness. Conclusions: Non-transmural scar, mildly decreased tissue thickness, and steep gradients in tissue thickness represent the structural characteristics of the diastolic component of reentrant circuits in VT-HC in this porcine model and could form the basis for imaging criteria to define ablation targets in future trials.

Individualised and non-contact post-mortem interval determination of human bodies using visible and thermal 3D imaging.

Determining the time since death, i.e., post-mortem interval (PMI), often plays a key role in forensic investigations. The current standard PMI-estimation method empirically correlates rectal temperatures and PMIs, frequently necessitating subjective correction factors. To overcome this, we previously developed a thermodynamic finite-difference (TFD) algorithm, providing a rigorous method to simulate post-mortem temperatures of bodies assuming a straight posture. However, in forensic practice, bodies are often found in non-straight postures, potentially limiting applicability of this algorithm in these cases. Here, we develop an individualised approach, enabling PMI reconstruction for bodies in arbitrary postures, by combining photogrammetry and TFD modelling. Utilising thermal photogrammetry, this approach also represents the first non-contact method for PMI reconstruction. The performed lab and crime scene validations reveal PMI reconstruction accuracies of 0.26 h ± 1.38 h for true PMIs between 2 h and 35 h and total procedural durations of ~15 min. Together, these findings broaden the potential applicability of TFD-based PMI reconstruction.

Improving the visualization of fingermarks using multi-target immunolabeling.

The development of fingermarks is an important step in visualizing ridge patterns for individualization purposes. Immunolabeling can be applied to fingermarks to selectively and sensitively detect antigens in fingermarks, and can be used as a developing method to visualize fingermarks. In this study we investigated single (the detection of one antigen) and multiple targeting approaches (the detection of multiple antigens simultaneously) to improve fingermark development. The detection of dermcidin, an antimicrobial peptide, was used as the gold standard to compare single and multi-target detection of keratins, albumin and/or dermcidin. Single detection of dermcidin and albumin mostly resulted in clear ridge details and/or pore detection, whereas the single keratin detection resulted in a poor visualization of the fingermarks. The multi-target approach in which both dermcidin and albumin were targeted, resulted in improved fingermark development compared to single dermcidin detection. Therefore, we recommend the use of multi-target detection consisting of anti-dermcidin and anti-albumin when using immunolabeling as fingermark development technique. Additionally, the optimized multi-target approach was tested as a pre- and post-development technique in combination with powder dusting and cyanoacrylate fuming. Immunolabeling has not been implemented yet in forensic case work, however we expect that immunolabeling can be used to redevelop poorly developed and/or smudged fingermarks in the nearby future. Currently, an ongoing pilot-study is being conducted in collaboration with the Dutch police.

The compatibility of immunolabeling with STR profiling.

Immunolabeling is a technique, which has recently been introduced to enhance the quality of developed fingermarks and subsequently strengthen the evidential value. The effect of this method on subsequent DNA analysis, however, has not been explored yet. Therefore, the current pilot study aimed to determine whether STR profiling is possible after immunolabeling. Since immunolabeling involves washing steps which could reduce DNA quantities, the use of different fixatives including methanol, formaldehyde and universal molecular fixative (UMFIX) were investigated. STR profiles from the (immunolabeled) fingermarks were generated after four days and four weeks by a direct PCR method to enable comparison of relatively fresh and old fingermarks. The fingermarks were deposited on diverse forensically relevant substrates, including glass, metal and tile. STR profiles could be recovered for all tested fixatives with no significant difference in performance. However, the mean number of detected alleles was the highest when methanol was used for fixation. Furthermore, immunolabeling on aged fingermarks (4 weeks) was also possible, but the number of detected alleles showed a non-significant decrease. DNA could be recovered from deposits on all substrates, of which glass showed the highest mean number of detected alleles followed by metal and tile.

Bayesian analysis of depth resolved OCT attenuation coefficients.

Optical coherence tomography (OCT) is an optical technique which allows for volumetric visualization of the internal structures of translucent materials. Additional information can be gained by measuring the rate of signal attenuation in depth. Techniques have been developed to estimate the rate of attenuation on a voxel by voxel basis. This depth resolved attenuation analysis gives insight into tissue structure and organization in a spatially resolved way. However, the presence of speckle in the OCT measurement causes the attenuation coefficient image to contain unrealistic fluctuations and makes the reliability of these images at the voxel level poor. While the distribution of speckle in OCT images has appeared in literature, the resulting voxelwise corruption of the attenuation analysis has not. In this work, the estimated depth resolved attenuation coefficient from OCT data with speckle is shown to be approximately exponentially distributed. After this, a prior distribution for the depth resolved attenuation coefficient is derived for a simple system using statistical mechanics. Finally, given a set of depth resolved estimates which were made from OCT data in the presence of speckle, a posterior probability distribution for the true voxelwise attenuation coefficient is derived and a Bayesian voxelwise estimator for the coefficient is given. These results are demonstrated in simulation and validated experimentally.

Phosphorescence of thermally altered human bone.

Bone has photoluminescent characteristics that can aid the analysis of thermally altered human skeletal remains as part of the forensic anthropological investigation. Photoluminescence stands collectively for fluorescence and phosphorescence. Because the difference in lifetime between fluorescence and phosphorescence is usually in the range of nano- to microseconds, it is only possible to visually determine whether bone phosphoresces when the lifetime is long enough to be observed. For this study, a distinction was made between long-decay and short-decay phosphorescence. So far, it was unknown whether (thermally altered) human bone emits long-decay phosphorescence after being illuminated and, thus, whether phosphorescence contributes to the observed photoluminescence. If so, whether the observable phosphorescence is dependent on temperature, exposure duration, surrounding medium, bone type, skeletal element, and excitation light and could aid the temperature estimation of heated bone fragments. In this study, bone samples were subjected to heat in the range of from room temperature to 900 °C for various durations in either air or adipose as surrounding medium. In addition, different skeletal elements of a human cadaver were recollected after cremation in a crematorium. Both sample collections were illuminated with light of different bandwidths and visually inspected for phosphorescence and photoluminescence. The samples were scored by means of a scoring index for the intensity of long-decay phosphorescence and photographically documented. The results show that thermally altered human bone fragments do phosphoresce. The observed phosphorescence is more dependent on temperature than on exposure duration, surrounding medium or skeletal element. Of the used wavelength bands, ultraviolet light provided the most temperature-related information, showing changes in both phosphorescence intensity and emission spectrum. Long-decay phosphorescence and fluorescence with short-decay phosphorescence coincide; however, there are also temperature-dependent differences. It is therefore concluded that phosphorescence contributes to the observable photoluminescence and that the visibly observable phosphorescent characteristics can aid the temperature estimation of cremated human skeletal fragments.

The applicability of forensic time since death estimation methods for buried bodies in advanced decomposition stages.

Estimation of the postmortem interval in advanced postmortem stages is a challenging task. Although there are several approaches available for addressing postmortem changes of a (human) body or its environment (ecologically and/or biochemically), most are restricted to specific timeframes and/or individual and environmental conditions. It is well known, for instance, that buried bodies decompose in a remarkably different manner than on the ground surface. However, data on how established methods for PMI estimation perform under these conditions are scarce. It is important to understand whether and how postmortem changes are affected under burial conditions, if corrective factors could be conceived, or if methods have to be excluded for respective cases. We present the first multi-methodological assessment of human postmortem decomposition carried out on buried body donors in Europe, at the Amsterdam Research Initiative for Sub-surface Taphonomy and Anthropology (ARISTA) in the Netherlands. We used a multidisciplinary approach to investigate postmortem changes of morphology, skeletal muscle protein decomposition, presence of insects and other necrophilous animals as well as microbial communities (i.e., microbiomes) from August to November 2018 associated with two complete body exhumations and eight partial exhumations. Our results clearly display the current possibilities and limitations of methods for PMI estimation in buried remains and provide a baseline for future research and application.