Forensic pilot application of rehydrating solutions on human cadaveric skin: what are the effects on hemorrhagic infiltrates?

The microscopic evaluation of hemorrhagic infiltrates is crucial in forensic diagnostics, but it proves challenging in corificated and mummified cadavers. In these cases, pre-treatment with rehydrating solutions is recommended, although their effects on the hemorrhagic infiltrate are not well understood. In this pilot study, we microscopically investigated the effect of two different rehydrating solutions-Sandison's solution and fabric softener-on well-preserved human cadaveric skin samples taken from areas affected by an ecchymotic lesion, comparing them with direct fixation in formalin. Specifically, we examined the topographic distribution of the hemorrhagic infiltrate in each layer of the skin by assigning a semi-quantitative score, conducted mutual comparisons, and performed statistical analysis. Histologically, compared to direct fixation in formalin, a slight and statistically non-significant reduction in the hemorrhagic infiltrate was observed in samples pre-treated with fabric softener. On the other hand, a more pronounced and statistically significant decrease in scores was observed in samples pre-treated with Sandison's solution. This effect is likely due to the fact that Sandison's solution, due to its components, exerts an osmotic effect, partially inducing osmotic lysis of red blood cells. Overall, extensive areas of hemorrhagic infiltrates were preserved, although to a lesser extent, while smaller foci were markedly reduced, sometimes even disappearing. The findings suggest that Sandison's solution has a detrimental effect on cutaneous hemorrhagic infiltrates, emphasizing the importance of being cautious and conducting dual sampling, using both formalin and a rehydrating solution, for forensic examination of mummified or corificated skin samples.

Elastic fiber degradation as a possible indicator for PMI estimation on mummified and corified skin: a pilot study.

Mummified and corified bodies are particularly complex scenarios to investigate, starting from identifying the post-mortem interval (PMI), even more so in indoor environments. In these bodies, the skin has the peculiar feature to resist for a long time. Among its components, there are elastic fibers, which are characterized by intrinsic resistance to post-mortem degenerative phenomena. Starting from these considerations, we investigated microscopically the persistence, detectability, and changes of elastic fibers in the skin of mummified and corified bodies with different known PMI. The aim was to evaluate whether they could provide an additional tool to aid in PMI estimation in these cases. Therefore, we collected skin samples from mummified or corified bodies found in a domestic environment with different known PMI, as well as from corified bodies that had been exhumed after 11 years of burial. Histochemical staining specific for elastic fibers, namely, Weigert's resorcin fuchsin, showed their prolonged persistence and a progressive and different degradation between mummified and corified skin as a function of PMI. Moreover, on the whole, we observed greater preservation of elastic fibers in mummified skin than in corified one at the same PMI. Therefore, histological analysis of elastic fibers in mummified and corified skin may help to provide valuable aid in estimating PMI, especially in those particular cases where more reliable alternatives are lacking.

Use of cadaveric vitreous humor as an innovative substrate for diatoms research and forensic diagnosis of drowning.

The search for diatoms is the test that most of all may contribute to the forensic diagnosis of drowning. Diatoms can be extracted through different methods, which are all complex and long. In this preliminary study, we assessed human vitreous humor as an innovative substrate on which to research diatoms. Sampling and analyses were performed on 3 groups of 10 corpses each. The first one was composed of drowned victims. The second one of victims of traffic accidents with subsequent projection in water; based on the severe traumatic injuries reported at autopsy, we considered such victims as ideally assimilable to bodies immersed in water postmortem. The third group was composed of subjects who died for natural causes and without any relationship with water. The vitreous humor was centrifugated and cytocentrifuged and spotted on two different histological slides: one was left blank and the other one was assessed with hematoxylin and eosin staining. Microscopic observation successfully revealed diatoms in all cases of the first group; in the second group diatoms were not detected in 6 cases; in the third group, no diatoms at all were observed. Diatoms were always qualitatively and quantitatively consistent with those found in the corresponding viscera and waters, which had been investigated through classical acid digestion. Our analyses have demonstrated that the vitreous humor behaves in a completely similar way to the other viscera in cases of drowning. Although further investigations are necessary, vitreous humor has proved to be an innovative, suitable, and reliable substrate for the forensic research of diatoms.

Technical note: A comparison between rehydrating solutions in the pretreatment of mummified and corified skin for forensic microscopic examination.

Microscopic examination of mummified or corified skin may be of extreme importance for forensic purposes. However, standard histological samples in these cases are low-end, and preparation is burdened by several problems and so are diagnostic results: an improvement of these types of specimens is therefore advantageous. This study aims to identify the best performing rehydration solution among a fabric softener, a body lotion, and Sandison's rehydrating solution. Samples of skin undergoing mummification or corification were collected from 25 corpses and each sample was divided into 4 fragments: one of these fragments was directly fixated in 4% formalin, one was previously treated with a tissue softener, another one was previously treated with a body lotion, and the last one was treated with Sandison's solution. After 72 h, the pretreated samples were post-fixated in 4% formalin and then prepared for standard histological examination staining the histological slides with hematoxylin-eosin and Masson's trichrome. At the microscopic examination, samples directly fixated in formalin were characterized by usual marked structural alterations and altered stainability, typical of such dry tissues. Vice versa, those previously treated appeared to be better-preserved even though with different improvement levels: body lotion made a medium-low-grade restoration of the tissues, and fabric softener a high-grade restoration, while Sandison's rehydrating solution produced an optimal grade restoration. Sandison's rehydrating solution was confirmed to be the best rehydrating substance for mummified and corified skin. Fabric softener could be, however, considered a valid substitute, being productive of high-grade microscopic yield.

Postmortem Diagnosis of Invasive Disseminated Aspergillosis After Tongue Piercing.

ABSTRACT: Severe fungal infections caused by highly invasive fungi such as Aspergillus are not easy to diagnose and often have a poor prognosis. In these cases, the nonspecific symptoms may make clinical diagnosis challenging, and consequently, the autopsy and postmortem histological investigations acquire a crucial role. We report the case of a young man in good health who died of septic shock 3 weeks after having had a tongue piercing. Intravitam investigations did not identify the etiology of the rapidly fatal infectious condition. The autopsy revealed flaccid organs of uniformly diminished consistency with abscesses and granulomatous foci with central necrosis. Histological examination showed the presence of septate mycotic hyphae, with a dichotomous 45-degree bifurcation, typical for Aspergillus , in all the examined organs, including the tongue. The molecular identification confirmed the presence of Aspergillus fumigatus. The observed macroscopic framework and the laboratory findings made it possible to diagnose pseudomembranous invasive tracheobronchial aspergillosis and to attribute the death to fatal invasive disseminated aspergillosis. The consistency and concordance of all the findings in our possession led us to suspect the practice of piercing as the triggering cause of the man's pathology.

Technical note-stabilization of cadaveric corified and mummified skin thanks to prolonged temperature.

Microscopic study and interpretation of skin samples obtained from mummified/corified cadavers request the adoption of special laboratory techniques in order to avoid that the histological samples may detach from the slide during staining processes. We have assessed an experimental and extended drying of corified and mummified skin samples in order to evaluate an eventual improvement after staining procedures. Samples of skin undergoing different post-mortem putrefactive phenomena-from parchment-like to mummification-were collected from 16 corpses and divided in halves: one half was fixated in buffered formalin while the other half was previously treated with Sandison's rehydrating solution. The so-obtained sections were subsequently dried for one night at 37 °C and stained with Hematoxylin & Eosin staining and Masson's Trichrome staining according to Goldner. At microscope observation, 69% of the dyed sections stained with H&E and 53% of the sections stained with Masson's Trichrome was lost during different steps of the coloring procedures. We then proceeded with a new cutting of the slides and decided to experimentally dry them for 5 days at a mean temperature of 37 °C: purpose of this procedure was to gradually remove the possible liquid component of the samples probably culpable of the detachment. With this modified procedure, all the sections resulted to be preserved in the following staining steps and showed, moreover, an improvement of the morphological structure, especially in those previously treated with Sandison's rehydrating solution. In those cases where a histological examination is requested on skin samples collected from corified or mummified cadavers, this technique is highly recommended in order to obtain microscopic slide of high quality.

A Brief Review of Scanning Electron Microscopy With Energy-Dispersive X-ray Use in Forensic Medicine.

Scanning electron microscopy with energy-dispersive x-ray (SEM/EDX) analysis is an investigation whose potential has become increasingly important in the field of forensic research and diagnosis. We present the procedure to perform a well-carried-out SEM/EDX analysis on corpses affected by different types of injuries, such as blunt force trauma, ligature strangulation, electrocution, sharp force trauma, gunshot wounds, and intoxication. After the areas of forensic interest have been macroscopically identified, the sampling can be performed in 2 different ways: apposition of the double-sided graphite tape on the damaged area or performing the excision of a biological sample. In both cases, a proper negative control sample is required. In all cases, SEM/EDX analysis can detect exogenous microtraces consistent with the types of injuries involved. In blunt force trauma, microparticles of different nature deriving from the contact of the blunt instrument with the victim may be observed; in sharp force trauma, metal microtraces (Fe, Cr, Al, Ti) can be identified. In ligature strangulation, exogenous microtraces may be found in the cutaneous furrow. In electrocution, it allows to identify the pathognomonic metal pattern (Cu, Zn, Fe) of the "electric mark." In gunshot wounds, the main applications regards the detection of metal particles (Pb, Ba, Sb) of gunshot residues. Finally, in the analysis of intoxicants, it may identify traces of toxic substances. Thus, the authors conclude that SEM/EDX analysis can provide essential information to assist in the medicolegal investigation of death.

Characterization With Scanning Electron Microscopy/Energy-Dispersive X-ray Spectrometry of Microtraces From the Ligature Mean in Hanging Mechanical Asphyxia: A Series of Forensic Cases.

The authors applied scanning electron microscopy with energy-dispersive x-ray spectrometry to the furrow derived from hanging means. The study was conducted with the purpose to detect possible extraneous microtraces, deriving from the ligature, that could have had an interaction with the cutaneous biological matrix, thanks to a transfert mechanism, in the proximities of the lesion.Fifteen cutaneous samples of the furrow and an equal number of fragments of graphite tape, directly positioned on the lesion produced by the ligature mean and used as a "conductor" of possible traces, were analyzed using scanning electron microscopy with energy-dispersive x-ray spectrometry.The research of microscopic traces on the furrow using this technique highlights extraneous traces leading to 3 main categories: natural fabrics, and synthetic and metallic materials, excluding possible environmental pollutants. The analysis, run on 7 hanging deaths, made available by the judicial authority, found a morphological and compositional compatibility with the traces found on the cutaneous furrow produced during hanging.The technique used in this study is innovative in the pathological-forensic field, and can be considered useful in clarifying and studying this typology of asphyxia leading to a specific ligature material, when missing, or attributing the cause of death to hanging when the furrow is not macroscopically obvious.

The taphonomy of blood components in decomposing bone and its relevance to physical anthropology.

OBJECTIVES: The variation and persistence of blood components, in particular red blood cells (RBCs), within bone tissue during the decomposition process, especially at the early stages and in different taphonomic conditions, has never been thoroughly investigated, regardless of the fact that knowing how blood survives or degrades within bone could be of help in solving many anthropological issues, such as trauma analysis and interpretation. MATERIALS AND METHODS: This research investigated the influence of time and taphonomy on the persistence and detectability of blood components in parietal bone fragments (of different post mortem periods and taphonomic conditions) through histological (Hematoxilin and Eosin, HE) and immunohistochemical (Glycophorin A, GYPA) analyses. RESULTS: The immunohistochemical investigation for GYPA showed the presence of RBCs under the form of erythrocyte debris or residues otherwise morphologically unidentifiable using only HE staining. Hence, while well-defined RBCs can be observed only in the first week of decomposition, afterward these structures can be detectable with certainty only by immunohistochemical analysis, which reveals discrete quantities of RBC residues also in dry bone (post mortem interval, or PMI, of 15 years), but not in archaeological samples, in which the greater PMI and the different taphonomic conditions together could be the answer behind such difference. DISCUSSION: This study highlights the usefulness and potential of immunohistochemical detection of GYPA in RBC investigation and gives a realistic idea of the persistence and detectability of erythrocytes in different osteological taphonomic conditions, in contrast to results reported by some authors in literature. Another important result concerns the detection of RBC residues in dry bone, which opens the way to the possible use of RBCs in trauma interpretation.

Could synovial fluid be a useful substrate for the forensic analysis of diatoms?

To date, synovial fluid has not been the subject of targeted analysis as a possible substrate to search for the presence of diatoms in the forensic context of drowning. However, its unique characteristics of production and isolation from the external environment could make it suitable for this purpose, similar to what has already been demonstrated in the literature for vitreous humor. By considering this, synovial fluid was analyzed in a specific case that came to our attention, where the coexisting signs of polytrauma and drowning were documented during autopsy, demonstrating a period of vitality during immersion. After a thin smear of the supernatant was obtained from the centrifugation of the synovial fluid sample, diatoms were successfully detected, consistent with those found in other organs and the water of the canal. The detection of diatoms in the synovial fluid was an objective finding, but its generalizability is limited because this was a pilot application. However, in cases where death by drowning is suspected and the body has multiple areas breached by trauma, the technique of analyzing diatoms in the synovial fluid could have great potential. Therefore, it is appropriate to further explore this technique in order to obtain more forensic evidence in such a setting.

Colorimetric Barium Detection of Gunshot Residues on Cadaveric Human Skin: A Pilot Application for Forensic Purposes.

Introduction: In this study we microscopically investigated, for the first time ever, the colorimetric detectability of barium of gunshot residues (GSR) on cadaveric human skin with gunshot wounds. Methods: For this purpose we used two different colorimetric techniques known in the literature, namely 0.2% sodium rhodizonate (Na-R-Ba 0.2%) and sodium rhodizonate in alcoholic environment (Na-R-Ba OH 0.2%). At the same time, we have also coupled it with scanning electron microscopy with energy dispersive X-ray (SEM/EDX) analysis and the colorimetric study for the detection of lead of GSR. These techniques were applied to 16 victims who died from gunshot injuries, as well as to a control group. Results: SEM/EDX demonstrated the presence of lead in all cases and barium in 11 of the 16 cases. The subsequent colorimetric technique with Na-R-Ba 0.2% did not show the barium of GSR in any case, unlike the Na-R-Ba OH 0.2% technique. This latter, in fact, has demonstrated the presence of this metal in 2 cases (18%). No microscopic case of false positive was recorded. Conclusion: The evidence obtained with Na-R-Ba OH 0.2% makes this method, applied here for the first time ever, worthy of further study. Meanwhile, although this technique can certainly be applied, it cannot be separated from the contextual colorimetric investigation for lead and the use of more sophisticated techniques.

Burial of piglet carcasses in cement: a study of macroscopic and microscopic alterations on an animal model.

Scarce experimental data exist describing postmortem effects of burial in cement. The scanty literature presents several case reports, but no experimental study. To perform a pilot study, the following experimental system was designed: 4 piglet corpses, who died of natural causes, were encased in concrete. After 1, 2, 3, and 6 months, a block was opened, and autopsy and microscopic analyses were performed. At the first month, initial putrefaction had started, and hindlegs were partly skeletonized. At the second month, both forelegs and hindlegs were partly skeletonized, and the abdomen and back showed advanced putrefaction. At the third month, the samples showed areas of mummification at the abdomen within a general context of initial putrefaction. At the sixth month, the sample showed wide adipocere formation. Histological findings revealed in some analyzed tissues (epithelium, dermis, adipose, and subcutaneous muscular tissues) a well-defined histological pattern even at 3 months after encasement in concrete: this means that microscopic changes may be delayed in concrete and that it may be worth performing histological analyses even in such kind of decomposed material.

Decomposition and entomological colonization of charred bodies - a pilot study.

AIM: To use forensic entomological approach to estimate the post mortem interval (PMI) in burnt remains. METHODS: Two experiments were performed in a field in the outskirts of Milan, in winter and summer 2007. Four 60-kg pigs were used: two for each experiment. One pig carcass was burnt until it reached the level 2-3 of the Glassman-Crow scale and the not-burnt carcass was used as a control. In order to describe the decomposition process and to collect the data useful for minimum PMI estimation, macroscopic, histological, and entomological analyses were performed. RESULTS: In the winter part of the experiment, the first insect activity on the burnt carcass began in the third week (Calliphora vomitoria) and at the beginning of the fourth week an increase in the number of species was observed. In the summer part, adult flies and first instar maggots (Phormia regina) appeared a few minutes/hours after the carcass exposure. Both in winter and summer, flies belonging to the first colonization wave (Calliphoridae) appeared on burnt and control pigs at the same time, whereas other species (Diptera and Coleoptera) appeared earlier on burnt pigs. CONCLUSION: In forensic practice, burnt bodies are among the most neglected fields of entomological research, since they are supposed to be an inadequate substratum for insect colonization. Entomological approach for PMI estimation proved to be useful, although further studies on larger samples are needed.

Lethal Bochdalek hernia in a three-year-old: pathological findings and medicolegal investigation in accusation of malpractice.

Diaphragmatic hernias can be a pitfall for paediatric diagnostics, especially in the cases of late presentation, which can also have medico-legal ramifications as exposed. A three-year-old boy was taken to a children's hospital after an episode of abdominal pain and vomiting of food. A physical examination proved normal, with mild ketonuria being found and he was discharged. Three months later for the same signs and symptoms, he was taken once more to hospital, where he arrived in a state of cardiac arrest and died. A lawsuit was begun against the doctors who had examined him three months earlier. The autopsy found herniation of the abdominal organs into the left pleural cavity through a defect in the left hemidiaphragm. The cause of death was identified as cardiac tamponade caused by mechanical compression of multiple herniated abdominal organs. The histopathological examination revealed marked atelectasis of the left lung, with non-expansion of 60-90% of the alveoli which suggested an acute mechanism that proved fatal, and the doctors were acquitted. The features of this disease and the possible difficulties in its diagnosis highlight the need for the utmost attention in differential diagnosis, even at an age where the discovery of a diaphragmatic hernia is exceedingly rare.

Utility and diagnostic value of postmortem microbiology associated with histology for forensic purposes.

Nowadays, the diagnostic value of postmortem microbiological investigations is still a debated topic, but postmortem microbiology (PMM) remains a discipline with great forensic potential. To evaluate the usefulness and diagnostic-forensic value of postmortem microbiological cultures, it has been conducted a study on cadaveric material sampled during autopsy aiming to identify the correct cause of death. The study analyzed 45 cadavers subjected to judicial autopsy, divided into two groups based on the presence or absence of external or internal macroscopic autopsy signs suggesting infectious pathology. In the same cases, both the microbiological and conventional histological investigations have been simultaneously carried out. From the investigations, mono-bacterial, mono-fungal, mixed and negative cultures were observed. In mono-species microbiological growth, the histological epicrisis confirmed an infectious cause of death due to the presence of signs of acute infection with an aggressive infectious agent. In cases where growth was mixed, it was possible to distinguish between simple postmortal contamination and perimortem colonization. Finally, in some cases where the microbiology was negative, this has been essential in highlighting signs of a vital reaction to viral or parasitic infection. The joint and integrated evaluation of the laboratory results made it possible to correctly understand even those peculiar situations in which the PMM results alone would not have been significant. These methods, when combined, constitute an optimal forensic approach for the identification of the real cause of death and thus reduce the number of unsolved cases.

Histotopography of haemorrhagic infiltration in the hanging cutaneous furrow: Where to look for haemorrhagic infiltration in hanging.

The forensic evidence of hanging is based on the autopsy demonstration of the traces left by the noose or the ligature on the neck, as well as on the histological assessment of the hanging mark vitality. However, the specific topography of haemorrhagic infiltration in the context of the cervical damaged tissues involved in hanging is not known. We carried out an extensive microscopic examination to identify if haemorrhagic infiltration in hanging appeared in specific topographic locations that could have been considered as elective. From 102 victims of suicidal hanging, a skin fragment was sampled from the maximum compression area of the sulcus, including the skin portions immediately above and below it. The sampling was also extended to collect the subcutaneous adipose tissue and part of the striated muscle. A standard histological examination was performed on all the samples, and hematoxylin and Eosin, Weigert's resorcin-fuchsin and Goldner's Masson trichrome staining were performed. In all the cases assessed, the microscopic examination allowed the detection of haemorrhagic infiltration mainly in the deepest areas under the sulcus and especially in the deep dermis and at the transition point between the dermis and the subcutaneous adipose tissue, as well as in the context of its supporting connective tissue. Such areas could therefore be considered as regions in which the presence of haemorrhagic infiltration is more likely to be demonstrated. Accordingly, we recommend performing sampling similar to ours and focusing the search for haemorrhagic infiltration as suggested.

Visceral Microscopic Pattern From Suicidal Ingestion of Professional Lysoform® With Delayed Death.

Lysoform® in the formulation of professional detergent is widely used in several fields, whereas its suicidal ingestion is an unusual occurrence. Therefore, the biological signs of this fatal poisoning remain unclear and elusive, similarly to the histological lesions induced by its main constituent, which is benzalkonium chloride (BZK). Furthermore, since all the deaths that has been reported in the literature occurred immediately, microscopic pictures of BZK lethal toxicity in subjects with prolonged survival have never been reported to date. Specifically, this brief communication reports the unique case of a woman who ingested professional Lysoform® to commit suicide, for which she died two weeks later. The autopsy examination showed either local or systemic signs of caustic ingestion; moreover, the histological analysis showed clear cellular damage of lungs, heart, and kidneys. In our case, toxicological investigations were not authorized as they were no longer considered significant. In this framework, the histological examination has therefore assumed a fundamental role in investigating and demonstrating the lethal effects caused by the systemic dissemination of BZK, which would otherwise no longer be investigable. Therefore, in cases of substance intoxication with prolonged survival where forensic toxicological investigations may be no longer possible or feasible, the histological examination may be the only resource to successfully observe and demonstrate its lethal effects.

Histological microaggregates as a sign of systemic dissemination due to oral ingestion of povidone-iodine (Betadine®).

Povidone-iodine (PVP-I) in the formulation of Betadine® is widely used in several medical fields, whereas its suicidal ingestion is an unusual occurrence. Therefore, the biological signs of this fatal poisoning remain unclear and elusive, similarly to the histological lesions induced by PVP-I. In fact, there are no forensic articles concerning the histological signs of PVP-I acute poisoning. Specifically, this short communication reports the unique case of a man who ingested 125 ml of Betadine® 10% to commit suicide, for which he died three days later. The autopsy examination showed either local or systemic signs of caustic ingestion; moreover, the histological analysis showed both intra- and extra-cytoplasmic amorphous and brownish microaggregates in almost all the organs. Histological and histochemical techniques resulted to be negative for the detection of physiological and known pigments (e.g., formalin, lipofuscin, hemosiderin). Likewise, such pigments were not associated neither with the man's diseases nor with his medications. Therefore, the authors supposed that they were povidone-iodine microaggregates, which have been deposited in the organs through the blood circulation. In conclusion, the complete postmortem histological examination could allow the identification and the characterization of PVP-I microaggregates as evidence of systemic toxicity from Betadine®, when dealing with a forensic case of ingestion of povidone-iodine.

Release of metals from osteosynthesis implants as a method for identification: post-autopsy histopathological and ultrastructural forensic study.

Metal structures--especially of stainless steel, titanium and their alloys (biomaterials)--are widely used in orthopaedic practice and the subject of constant study in bioengineering and preventive medicine. This study presents the first experience of forensic research into the presence of permanent tissue variations around metal implants in various bone structures for the purpose of identification, with particular reference to skeletal remains or severely decomposed corpses in the absence of other identifying elements. The evaluation was conducted on 12 corpses who had undergone osteosynthesis intra-vitam, whose implants were still in place or had been removed, in comparison with five controls who had never undergone osteosynthesis. Bone fragments taken during autopsy were subjected to histopathological and scanning electron microscope-energy dispersive electroscopy examination in order to reveal and characterise any metal particles originating from osteosynthesis. The study enabled the discovery of intra-bone metal particles in tissues treated by osteosynthesis even in bone areas where the implants had been removed and even where there were no longer any radiological signs of their application. These results are therefore of considerable forensic importance, especially in the area of identification, providing a valid means of recognition beyond that of the well-established use of in situ metal implants.