Chemical investigation of biological trace evidence; toxicological screening of waste residues obtained from DNA extraction processes.

In a forensic scenario, if biological stains are found in very small quantities, these are usually collected for DNA analyses, considered essential for the forensic investigation and thus excluding possible investigations by other forensic disciplines as forensic toxicology. We developed an experimental study to evaluate the feasibility of analyzing DNA extraction residues obtained from DNA extraction procedures to perform toxicological analysis, with the aim to extract both genetic and toxicological information without affecting or compromising the genetic sample and/or DNA extraction. DNA extraction from four blood samples (fortified with 5 molecules of interest with a final concentrations of 1 µg/mL, 100 ng/mL, 10 ng/mL and 5 ng/mL, respectively) were analyzed with QIAGEN QIAmp® DNA Mini kit. Three waste residues collected from the DNA extraction were analyzed for the toxicological investigation via Solid-Phase Extraction and High-Performance Liquid Chromatography-Tandem Mass Spectrometry analyses (Thermo Scientific™ TSQ Fortis™ II Triple-Quadrupole Mass Spectrometer). The analytical investigation revealed that our analytes of interest were detected in two different residues of the DNA extraction procedure, allowing both genetic and toxicological analyses without affecting the DNA identification. At last, the experimental protocol was applied to a hypothetical case, with encouraging results and allowing the identification of our molecules of interest.

A Forensic Case of Suicide Ingestion of Paraquat Herbicide: New Histological Insights and Revision of the Literature.

ABSTRACT: Paraquat (PQ) is one of the most widely used herbicides in the world, and poisoning is generally associated with accidental, suicidal, or homicidal events. Therefore, in the forensic context, PQ could be in various ways involved as a possible cause of death of a subject. However, even though its systemic toxicity is known, the biological effects exerted on individual viscera have been explored only to some extent, especially in case of victim's survival. Therefore, a case concerning a suicidal ingestion of PQ with survival of 3 days was deemed of interest. Clinical toxicological analyses confirmed acute PQ intoxication, and after the death of the victim, an autopsy was performed showing local and systemic signs of ingestion of a caustic substance. Histologic examination revealed marked cellular damage to the major viscera, particularly the gastroesophageal tract, liver, kidneys, and lungs, with initial alveolar fibrosis noted despite the patient's short survival. This finding represents a new element in the context of PQ lung injury, as it has not been previously documented in the literature. Thus, histological findings in lethal intoxications after survival can reveal specific peculiarities still unknown and, therefore, assume transversal relevance not only at forensic but also clinical level.

Water-related atmospheric agents and solubility: two parameters of validation in toxicological screening on clothing worn by skeletal remains.

In forensic toxicology, when conventional matrices are no longer available, alternative matrices can be used to assess toxicological investigations. Clothes worn by skeletal remains may be a good unconventional matrix for toxicological analyses considering that they have absorbed decomposition fluids and blood from a body. We hypothesized a scenario in which a skeleton, wearing clothes, was discovered in an open environment. From this starting point, an experimental study was developed on different textiles (cotton, wool, and polyester) to evaluate whether water-related atmospheric agents and molecule solubility can largely influence the detection of molecules of toxicological interest on this specific matrix, together with the characteristics of different garments chosen. The experimental study was performed on blood spots, previously spiked with 6-monoacetylmorphine and morphine, accurately placed on different textiles and washed with different quantities of deionized water adjusted at pH 5.6 with formic acid to simulate different rainfall conditions. Toxicological analyses were performed via Solid-Phase Extraction and High-Performance Liquid Chromatography-Tandem Mass Spectrometry analyses (Thermo Scientific™ TSQ Fortis™ II Triple-Quadrupole Mass Spectrometer). From the experimental study morphine could not be detected on 100% cotton and 100% wool fabric after the passing of 500 mL of deionized water and in 100% synthetic polyester textile after washing with 250 mL of deionized water. In conclusion, when toxicological analyses are carried out on unconventional matrices as textiles worn by corpses exposed to different environmental conditions, it is of great importance, in using such substrates as evidence for the presence of molecules of toxicological interest, to evaluate chemical-physical characteristics of each analyte under investigation in order to correctly interpret the toxicological data obtained.

A forensic case of abdominal cocoon syndrome.

The term "cocoon syndrome" defines a sclerosing encapsulating peritonitis (SEP) that involves a chronic fibrotic inflammatory reaction of the parietal peritoneum and of the viscera leading to a complete sclerosis. The cocoon that is formed causes an incarceration of the intestinal loops with severe complications leading to high mortality. We are presenting the case of a 15-year-old young man that underwent surgery for appendectomy and that was evaluated for having a regular abdominal state. During the post-surgery period, however, several episodes of intestinal occlusion required further surgical interventions leading to a right hemicolectomy. The presence of a fibrotic-adhesive ligneous peritonitis with blended intestinal loops, severely thickened walls, and intestinal scaring stenosis was observed during his second surgical operation. A stenosis of the colostomy led to a worsening of the vital signs of the young man with the onset of a cardiac failure and subsequent decease. Macroscopic autopsy examination and histological analysis confirmed the severe obstructive adhesive encapsulating abdominal context allowing to trace back the cause of death to a cocoon syndrome. Since no predisposing factor could be found, we hypothesized that this case could be characterized by an excessive peritoneal reactivity due to surgical appendectomy. Cocoon syndrome is a rare pathology, and its microscopic features are seldomly observed and could be underestimated. We present a directly observed case with a very substantial macroscopic and microscopic context.

A unique case of death by MDPHP with no other co-ingestion: a forensic toxicology case.

Synthetic cathinones are a class of psychoactive drugs that have become, in recent years, of quite common observation in medical and toxicological forensic field. MDPHP (3,4-methylenedioxy-α-pyrrolidinohexanophenone) belongs to this class of substances but lethal acute intoxication caused by this specific substance without other co-ingestions has never been described in literature. We are presenting the unique case of a fatal acute intoxication caused by MDPHP in a 48-year-old man carried to the A&E department of a big Milanese hospital where he suddenly deceased after brief time. Clinical records, autopsy examination, histological findings, and toxicological analysis, assessed via a Q-Exactive Orbitrap with a HPLC system and LC/MS-MS system, are discussed. This case report may represent the first case of this type of intoxication and might help in improving recognition and treatment of these particular cases.

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 diagnostics of assumed suicidal food anaphylaxis in prison: a unique case of anaphylactic death due to peach ingestion.

Suicidal ingestion of food which the victim is aware they are allergic to is an exceptional occurrence in the forensic field. To the best of our knowledge, no cases of suicidal food anaphylaxis have been reported to date. Therefore we present the first case described in the literature. A 30-year-old prisoner was found dead inside his cell with the remains of a peach remains next to his body, and a handwritten farewell note in his pocket. The autopsy revealed only non-specific findings, while laboratory investigations (serological, toxicological, histological, and immunohistochemical) played a pivotal role in determing the cause and manner of death. In particular, a high titer of both total and specific IgE antibodies was detected, as well as an increase of the tryptase level in cadaveric blood. Moreover, a massive concentration of salicylates was measured in the gastric contents. Microscopically, cellular residues characterized by a vegetal structure were observed in the gastric contents and elements suggestive of mast cells were detected in the glottis, lungs, and myocardium. The immunohistochemical investigation with anti-CD117 and anti-tryptase antibodies showed positivity for mast cells, some of which appeared degranulated. Such findings were entirely consistent with an acute systemic anaphylactic reaction triggered by allergy. Therefore, the prisoner's death was attributed to self-induced food anaphylaxis caused by the ingestion of peaches. This conclusion was achieved based only on circumstantial data, anamnestic information, autopsy findings, and multiple laboratory results. This integrated approach should be used to pursue a post-mortem diagnosis of anaphylaxis.

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 detection of metallic residues in skin stab wounds by means of SEM-EDS: A pilot study.

The morphological analysis of stab wounds may often not be accurate enough to link it with the type of wounding weapon, but a further evaluation may be performed with the search for metallic residues left during the contact between the instrument and the skin. In this study, Scanning Electron Microscopy-Energy Dispersive Spectroscopy (SEM-EDS) was applied to the study of cadaveric stab wounds performed with kitchen knives composed of iron, chromium and nickel, in order to verify the presence of metallic residues on the wound's edge. Two groups of 10 corpses were selected: group A, including victims of stab wounds and a control group B (died of natural causes). Samplings were performed on the lesions and in intact areas of group A, whereas in group B sampling were performed in non-exposed intact skin. Samples were then analysed with optical microscopy and SEM-EDS. In group A, optical microscopic analysis showed the presence of vital haemorrhagic infiltration, while SEM-EDS showed evidence of microscopic metal traces, isolated or clustered, consisting of iron, chromium and nickel. Moreover, in two cases organic residues of calcium and phosphate were detected, as a probable sign of bone lesion. Control samples (group A in intact areas and group B), were negative for the search of exogenous material to optical microscopy and SEM-EDS. The results show the utility and possible application of the SEM-EDS in theidentification of metallic residues from sharp weapons on the skin.

Acute vertical deceleration injury: A case of cranial impalement.

In forensic science and in modern times, impalement is a rare event. When this occurrence involves the head, it is even more unusual since this region is very small compared to the rest of the body. Although impalements are rare, they represent scenarios that must be treated with great care by forensic pathologists. They may involve a mixture of blunt and penetrating wounds, and the injuries observed at autopsy must be interpreted correctly. For this reason, if possible, the interconnection between the body and the impaling object should be maintained until the autopsy table. In this report, it is presented a case of cranial penetrating trauma that occurred after a suicidal fall from a great height with impact on the railing below, by reporting both the on-site scene inspection and autopsy data. The critical forensic issues that may arise in the practical management of such cases are discussed, starting from the fact that there is still no clear and unambiguous definition of impalement.

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.

Is It Correct to Consider Caustic Ingestion as a Nonviolent Method of Suicide? A Retrospective Analysis and Psychological Considerations.

BACKGROUND: Suicide methods chosen by victims are particularly critical in suicide risk research. To differentiate suicide deaths, it is usual to categorize them as violent and nonviolent depending on the detrimental method chosen by the victims. Caustic ingestion, for example, is traditionally considered as a nonviolent suicide method. It results in severe consequences for the human body and it is associated with high levels of lethality. METHODS: In this study, we retrospectively analyzed suicides that occurred between 1993 and 2021 in Milan (Italy) and that underwent autopsy. We compared a sample of 40 victims that ingested caustic substances with a sample of 460 victims of other chemical ingestion, and a sample of 3962 victims from violent suicide. Univariate analyses and univariate logistic regression models were performed. Suicides from caustic poisoning were significantly older, had a higher mean number of diseases and were more affected by psychiatric diseases compared to other chemical ingestion victims. By contrast, caustic suicides, compared to violent suicides, had a more balanced gender ratio, a higher mean number of diseases, were more affected by psychiatric diseases, had a higher rate of complex suicides (more than one modality), and had victims who died more frequently inside instead of outside. In logistic regression models, age was the only feature differentiating caustic from other chemical ingestion suicides while the features differentiating caustic from violent suicides were gender, mean number of diseases and suicide place. CONCLUSIONS: Suicides by caustic ingestion showed substantial differences compared to violent suicides, with a higher severe profile. However, some differences were reported comparing caustic ingestion to other chemical ingestion as well. Thus, we argue whether it is more appropriate to differentiate the suicidal ingestion of caustics from both violent and nonviolent suicide methods.

Applications of microbiology to different forensic scenarios - A narrative review.

In contrast to other forensic disciplines, forensic microbiology is still too often considered a "side activity" and is not able to make a real and concrete contribution to forensic investigations. Indeed, the various application aspects of this discipline still remain a niche activity and, as a result, microbiological investigations are often omitted or only approximated, in part due to poor report in the literature. However, in certain situations, forensic microbiology can prove to be extremely effective, if not crucial, when all other disciplines fail. Precisely because microorganisms can represent forensic evidence, in this narrative review all the major pathological forensic applications described in the literature have been presented. The goal of our review is to highlight the versatility and transversality of microbiology in forensic science and to provide a comprehensive source of literature to refer to when needed.

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.

Toxicological analysis in victims of sexual and domestic violence: A retrospective study of a 3-year period (2018-2020).

This manuscript presents an epidemiological investigation carried out on abuse victims who accessed the Sexual and Domestic Violence Service (SVS&D) of IRCCS Ca' Granda in Milan, Italy. The focal point of this research was the detection of alcohol, prescription medications, and illicit substances in victims who solicited help from the SVS&D center between 2018 and 2020. Over this three-year span, biological samples of blood and urine were procured from 207 victims, out of a patient pool of 2470. All collected samples were analyzed via High Performance Liquid Chromatography - Tandem Mass Spectrometry (HPLC-MS/MS) and Gas Chromatography - Mass Spectrometry (GC-MS). Toxicological examination results demonstrated that 43% of the cases tested positive for substances in 2018, 39% in 2019 and 60% of the cases in 2020. Overall, 45% of the victims tested resulted positive to some substance over a 3-year period, equivalent to 3.6% of the overall cases (2470 victims). Substances of toxicological interest were detected in 104 samples (out of 377, corresponding to 27.6%) belonging to 94 patients. The most detected classes of drugs were stimulants, antidepressants, benzodiazepines and antipsychotics. Moreover, BAC (Blood Alcohol Concentration) indicated positivity in 25 cases (out of 184 cases analyzed - 14% of positive cases). Based on this study's findings, we recommend broadening the range of substances evaluated in drug-facilitated sexual assaults and establishing standardized protocols for both national and international implementation. Implementing procedures would significantly enhance forensic support provided to victims of abuse seeking healthcare services post-incident.

Papaver somniferum in seventeenth century (Italy): archaeotoxicological study on brain and bone samples in patients from a hospital in Milan.

In this paper, we present the results of toxicological analyses of preserved brain tissue and bone samples from the remains of the seventeenth century patients of the Ospedale Maggiore, the main hospital in Milan and one of the most innovative hospitals in Europe from the Renaissance period. Beneath it, the crypt functioned as the burial place for the deceased of the hospital. In this multidisciplinary study of the remains, toxicological analyses in particular were performed with HPLC-MS/MS on different biological samples from nine individuals. Anthropological, paleopathological, histological, radiological examinations and radiocarbon dating were also carried out. As a result, archeotoxicological analyses revealed the presence of codeine, morphine, noscapine and papaverine, derived from Papaver somniferum, a plant present in the hospital pharmacopeia used as a narcotic, analgesic, astringent, coagulant, and antitussive agent. Such analyses have shed light on the pharmacological therapies administered to the patients near the time of death and have implemented our knowledge of medical treatment and drug administration in the 1600's.

Postmortem forensic toxicology cases: A retrospective review from Milan, Italy.

We are presenting a study on 136 cases performed in a 2-year period (2018-2019) at the Bureau of Legal Medicine of the University of Milan for which toxicological analyses were requested and we are making a detailed interpretation of clinical records and discussing toxicological results from each case included in the study. Total number of autopsies was 1323 and in 10.3% of the cases, toxicological analyses were requested to obtain further information. Analyses were assessed with High-Performance Liquid Chromatography-Mass Spectrometry system and Gas Chromatography-Mass Spectrometry analyses. Additionally, Blood Alcohol Concentration and detection of volatile substances were obtained with Head Space-Gas Chromatography-Mass Spectrometry system. From these analyses, 101 cases out of 136 provided positive results (74.3%). Main substances detected were cocaine, diazepam, morphine, and ethanol. The most representative profiles of individuals that emerged from this study were: a Caucasian male, age 41-50, that died for cocaine acute intoxication or was killed; a Caucasian male or female with a range-of-age of 31-50 deceased for simple suicide caused by acute intoxication or by complex suicide caused by acute intoxication and suffocation; and finally, a Caucasian male with a range-of-age 21-40 that died in a car accident without any toxicological evidence. From the results, acute intoxication at the time of death was confirmed in 54 cases and in 57 cases the toxicological analyses helped in the determination of the cause and manner of death. From this study, the importance of toxicological data among forensic sciences is confirmed.