Silvia Damiana Visonà

Evaluation of benzodiazepines and zolpidem in nails and their stability after prolonged exposure to chlorinated water

&NA; The study aims the development and validation of a LC–MS/MS method for the identification and quantification of benzodiazepines and zolpidem in nails as alternative keratinized matrix to hair in long‐term monitoring of anxiolytic and hypnotic drugs. Both fingernail and toenail samples (1–2 mm) were collected by clipping the excess overhang of the nail from volunteers and from postmortem cases. They were washed twice with organic solvents, dried under nitrogen stream, pulverized, immersed in a methanol solution (internal standard: diazepam‐D5) and sonicated up to two hours. The solution was then direct injected in the LC–MS/MS system. Mass spectrometry was set in MRM mode, selecting two transitions for each substance. 32 analytes among benzodiazepines, metabolites and hypnotics were included in the list. The method fulfilled the internationally required criteria for validation. Limits of detection ranged from 0.03 pg/mg (zolpidem) to 13.1 pg/mg (bromazepam). 9 subjects under therapy were positive at 7 different benzodiazepines and/or metabolites (lorazepam, desalkylflurazepam, bromazepam, diazepam, alprazolam, lormetazepam and prazepam), while 5 molecules were measured in 4 postmortem cases (diazepam, desmethyldiazepam, delorazepam, 7‐aminoclonazepam and zolpidem). In vitro experiments on eight authentic samples suggested that benzodiazepines in nails are influenced by the prolonged exposure to chlorinated water.

Diagnosis of electrocution: The application of scanning electron microscope and energy-dispersive X-ray spectroscopy in five cases

INTRODUCTION Deaths from electricity, generally, do not have specific findings at the autopsy. The diagnosis is commonly based on the circumstances of the death and the morphologic findings, above all the current mark. Yet, the skin injury due to an electrocution and other kinds of thermal injuries often cannot be differentiated with certainty. Therefore, there is a great interest in finding specific markers of electrocution. The search for the metallization of the skin through Scanning Electron Microscope equipped with Energy Dispersive X-Ray Spectroscopy (EDS) probe is of special importance in order to achieve a definite diagnosis in case of suspected electrocution. MATERIALS AND METHODS We selected five cases in which the electrocution was extremely likely considering the circumstances of the death. In each case a forensic autopsy was performed. Then, the skin specimens were stained with Hematoxylin Eosin and Perls. On the other hand, the skin lesions were examined with a scanning electron microscope equipped with EDS probe in order to evaluate the morphological ultrastructural features and the presence of deposits on the surface of the skin. RESULTS The typical skin injury of the electrocution (current mark) were macroscopically detected in all of the cases. The microscopic examination of the skin lesions revealed the typical spherical vacuoles in the horny layer and, in the epidermis, the elongation of the cell nuclei as well as necrosis. Perls staining was negative in 4 out 6 cases. Ultrastructural morphology revealed the evident vacuolization of the horny layer, elongation of epidermic cells, coagulation of the elastic fibers. EDS-MICROANALYSIS In the specimens collected from the site of contact with the conductor of case 1 and 2, the presence of the Kα peaks of iron was detected. In the corresponding specimens taken from cases 2, 4, 5 the microanalysis showed the Kα peaks of titanium. In case 3, titanium and carbon were found. CONCLUSIONS In the suspicion of electrocution, the integrated use of different tools is recommended, including macroscopic observation, H&E staining, iron-specific staining, scanning electron microscopy and EDS microanalysis. Only the careful interpretation of the results provided by all these methods can allow the pathologist to correctly identify the cause of the death. Particularly, the present study suggests that the microanalysis (SEM-EDS) represents a very useful tool for the diagnosis of electrocution, allowing the detection and the identification of the metals embedded in the skin and their evaluation in the context of the ultrastructural morphology.

Diagnosis of sudden cardiac death due to early myocardial ischemia: An ultrastructural and immunohistochemical study

The aim of this post-mortem ultrastructural and immunohistochemical study is to explore the characteristics of acute myocardial ischemia in the context of sudden death, using the combination of two different methods, both more insightful than ordinary histology. Transmission electron microscopy and immunohistochemistry, in addition to the traditional histology, were applied to study human heart specimens collected during forensic autopsies. The whole series was sub-grouped into cases (n=17) and controls (N=10). The control group consisted of unnatural death with a short agonal period (immediately lethal injuries). Heart samples of the two cohorts of subjects were prepared for electron microscopy. On the other hand, each specimen, formalin fixed and paraffin embedded, was stained with haematoxylin and eosin and immunoreacted with the following primary antibodies: anti-Fibronectin, anti- Connexin-43, anti-npCx43 (dephosphorylated form of Connexin43), anti-Zonula occludens-1. Immunopositivity for each marker in the myocardium was semi-quantitatively graded. Electron microscopy revealed a number of interesting differences, statistically significant, between acute myocardial ischemia and controls, regarding the morphology of nucleus, mitochondria and intercellular junctions. By immunohistochemistry, fibronectin was found to be increased in the extracellular matrix of the acute myocardial ischemia cases, with a statistically significant difference compared to the controls. Connexin 43 staining disclosed a slight increase (not statistically significant) in the cytoplasm of acute myocardial ischemia cases compared to the controls, whereas no significant differences were seen between cases and controls at intercellular junctions. npCx43 showed an evident difference of intensity and pattern (even though not statistically significant) in cases compared to controls and overall this difference was more evident in the cytoplasm. Zonula occludens 1, described as an important marker for functional modification of cardiac muscle fibers, resulted negative or very weak in the vast majority of both cases and controls. The present study attempts to simultaneously apply electron microscopy and immunohistochemistry, in order to figure out the morphological changes that might lead to pathological processes underlying the sudden, unexpected death due to acute myocardial ischemia, and consequently to find useful diagnostic markers of very early ischemic injury. Both methods showed significant differences between acute myocardial ischemia and controls, regarding, overall nuclei, mitochondria, and intercellular junctions.

Immunophenotypic analysis of the chronological events of tissue repair in aortic medial dissections

Acute medial dissection of aorta can occur in the context of a sudden and unexpected death. For medico-legal reasons it is important to estimate as accurately the histological age of dissections. We evaluated the additional value of a systematic application of immunohistochemistry, compared with conventional histology only, in determining chronological steps of injury and repair. Thirty two paraffin embedded specimens of aortic dissection were retrospectively allocated to one of four defined stages: acute (I), subacute (II), early organizing (III) and scarring (IV) using Hematoxylin and Eosin and Elastica van Gieson stained sections. Subsequent immunohistochemically staining was performed with the following markers: (myeloperoxidase (neutrophils), citrullinated-Histone 3 (neutrophil extracellular traps), CD68 (macrophages), CD3 (T-cells), CD31 and CD34 (endothelial cells), and smooth muscle actin. Immune stained sections were scored semi-quantitatively. Histologically, five cases were identified as stage I, 16 as II, 7 as III and 4 as IV. Additional immunostaining for smooth muscle cells and endothelial cells altered the classification in 25% of cases (all in groups II and III). Immunostaining and semi-quantitative grading of involvement of neutrophils, macrophages and NETs also provided specific distribution patterns over the 4 age categories, including unexpected involvement of the peri adventitial fat tissue. In conclusion, it appears that semi-quantitative immunohistochemistry of resident vascular wall cells, inflammatory cells and NETS represents a useful adjunct in detailed histopathological grading of the chronological age of aortic dissections.

Death of a seven-month-old child in a washing machine: a case report

The authors present a case which brings out a unique modality of child homicide by placing the baby in a washing machine and turning it on. The murder was perpetrated by the baby’s mother, who suffered from a serious depressive disorder. A postmortem RX and then a forensic autopsy were performed, followed by histologic examinations and toxicology. On the basis of the results of the autopsy, as well as the histology and the negative toxicological data, the cause of death was identified as acute asphyxia. This diagnosis was rendered in light of the absence of other causes of death, as well as the presence of typical signs of asphyxia, such as epicardial and pleural petechiae and, above all, the microscopic examinations, which pointed out a massive acute pulmonary emphysema. Regarding the cause of the asphyxia, at least two mechanisms can be identified: drowning and smothering. In addition, the histology of the brain revealed some findings that can be regarded as a consequence of the barotrauma due to the centrifugal force applied by the rotating drum of the washing machine. Another remarkable aspect is that we are dealing with a mentally-ill assailant. In fact, the baby’s mother, after a psychiatric examination, was confirmed to be suffering from a mental illness—a severe depressive disorder—and so she was adjudicated not-guilty-by-reason-of-insanity. This case warrants attention because of its uniqueness and complexity and, above all, its usefulness in the understanding of the pathophysiology of this particular manner of death.

Noncompaction cardiomyopathy in Hirschsprung's disease: a case report

Noncompaction cardiomyopathy is a rare disorder, often associated with cardiac and noncardiac malformations. Hirschsprungs disease, a well-known aganglionosis, is associated with congenital heart diseases and has been reported to be due to impairment migration and differentiation of neural crest cells. Here, we present an 8-month-old male infant who died for cardiogenic shock after surgical resection of the involved bowel segment. The child was affected by both noncompaction cardiomyopathy and Hirschsprungs disease, two entities which can share a common neural crest-derived etiology.

Sudden, unexpected death of a 15-year-old boy due to pancarditis: A case report and possible etiopathogenesis.

Background:Generally, rheumatic heart disease is, today, sporadic in developed countries, even though it continues to be a major health hazard in the developing ones. It is also a very rare cause of sudden unexpected death. We report a case of a 15-year-old boy who suddenly died at home. Since 3 days he had presented fever and chest pain. The family physician had diagnosed bronchitis and treated the boy with amoxicillin. Methods:Seven hours after death, a forensic autopsy were performed . Before the autopsy, anamnesis and some circumstantial data were collected from the boys parents. During the autopsy, samples for histological, toxicological and molecular examinations were collected. The samples for the histology (brain, hypophysis, heart and pericardium, lungs, spleen, liver, kidney, adrenal glands) were formalin fixed and paraffin embedded. Each section was stained with Hematoxylin-Eosin. Immunostaining was also performed, with anti-CD 68, anti-CD3, anti-CD 20, anti-myeloperoxidase. Microbiological cultures were performed on cardiac blood, myocardium, pericardial effusion and cerebrospinal fluid samples collected during autopsy. Blood specimens were also processed through PCR, in order to reveal the presence of Enteroviruses, Chickenpox virus, Epstein Barr virus. Also chemical-toxicological examinations for the detection of the main medications and drugs were performed on blood samples. Results:The anamnesis, collected before the autopsy, revealed an acute pharyngitis few weeks before. The autopsy, and the following histological and immunochemical examinations suggested an immunological etiology. The immunohistochemistry, showing a strong positivity of antiCD68 antibodies, integrated with clinical-anamnestic information, leads to hypothesize a rheumatic carditis. Conclusion:In light of this case, at least 3 main messages of great importance for the clinician can be deduced. First, an accurate anamnesis collected by the family physician could have led to the correct interpretation of the objective signs and the administration of an appropriate therapy. Second, a pharyngeal swab should be performed for cases involving sore throat in young children and adolescents, especially in cases involving repeated pharyngitis. Finally, apparently unremarkable findings revealed from objective examinations can be signs of a serious disease. Moreover, in some cases, these diseases can be lethal if they are not properly treated.

Neisseria meningitides Can Survive in Corpses for At Least Eleven Days.

Among the potential hazards of working in a mortuary and handling corpses, the risk of infectious disease acquisition is well-documented, and warrants attention. The main biological risk in this environment is due to infections caused by Mycobacterium tuberculosis, blood-borne hepatitis, and agents responsible for transmissible spongiform encephalopathies, such as variant Creutzfeld-Jakob disease. All these pathogens remain alive and are infectious postmortem. In addition, other pathogens present in cadavers, such as Neisseria meningitidis, are a potential source of infection during necropsy (Burton, 2003). Generally, it is believed that pathogens do not survive for more than a few minutes on environmental surfaces; however, N. meningitidis has been found to survive for up to 72 h on glass and metal surfaces (Tzeng et al., 2014). To date, there is a lack of data regarding the duration of survival of N. meningitidis in corpses, even though people who handle cadavers are commonly considered to be at risk of infection (Burton, 2003). During forensic examinations, deaths due to meningitis are often encountered. Recently, N. meningitides was detected in two corpses preserved at 4°C for 11 and 7 days, respectively, at our hospital. The first and more remarkable case regards a 28-year-old woman who died from fulminant meningitis. The anamnesis was negative until the day before she died. The woman presented to the Emergency Department with a high fever, shivers, nausea, and vomiting. The objective examination did not indicate any significant findings, except for abdominal tenderness and inflammation of the pharynx. Therefore, the patient was discharged. A few hours later, the woman returned to the same hospital with a higher fever (40°C) and persistent vomiting. Then, she was admitted to the Department of Internal Medicine. At the time of admission, blood samples were taken for microbiological testing, and antibiotic therapy (3 g of ampicillin and 2 g of cefotaxime) was administered. The clinical exams did not find anything remarkable except for the fever. At 2 h after admission, the woman presented with breathing difficulty, hypotension, diffuse petechiae, mild rigor nuchalis, and a temperature of 37.5°C. The patient was intubated, mechanical ventilation was enacted, and then she was brought to the Intensive Care Unit, where, 2 h later, she died. The microbiological test results, available after death, pointed out the presence of group C N. meningitidis in the blood. The day after, a medical autopsy was performed: macroscopically, diffuse petechiae and massive hemorrhage of the adrenal glands were observed. Bacterial cultures of the blood samples detected the growth of group C N. meningitidis. Eleven days after the womans death, the prosecutor ordered an additional autopsy in order to investigate the hypothesis of medical malpractice. Before the internal examination, using a carefully sterile technique, bacteriological samples were taken from blood (particularly from the neck vessels. Then, during the internal examination, samples of brain, liver, adrenal gland, bone marrow were collected using sterile instruments and containers. Briefly, blood (100 μL) was plated on selective Martin Lewis Agar (Becton Dickinson, Heidelberg, DE) and incubated in aerobic environment with 5% CO2 for 48 h. Specimens of other tissues were first inoculated in Brain Heart infusion Broth (Becton Dickinson, Sparks, US) and incubated in same conditions. After 24 h, 100 μL of broth were plated on Martin Lewis Agar and incubated in conditions of aerobic environment with 5% CO2. After 48 h of incubation we revealed growth of colonies on all the culture plates: in particular, on the plate inoculated with blood sample, we found more than 106 Colony Forming Units (CFU). At gram staining they appeared as Gram negative diplococci, with positive reaction for oxidase and catalase, suggesting Neisseria spp. The bacteria were identified as N. meningitidis using MALDI-TOF MS (Bruker Daltonics, Germany). Slide agglutination (Remel, US) showed reaction with serum against capsule polysaccharide of serogroup C. At the same time, we performed a molecular analysis on all the specimens. DNA extraction from clinical samples was carried out using the QIAamp DNA Mini Kit (Qiagen), according to manufacturer instructions. Purified DNA were kept at −80°C. Bacterial DNA was amplified both with 16S rRNA primers, a primer pair targeting ctrA gene for identification of N. meningitidis species, and six primers pairs targeting serogroup-specific capsule biosynthesis genes (A, B, C, X, Y, W135) of N. meningitidis. PCR was run with 35 cycles of 95°C for 30 s, 60°C for 1 min, 72°C for 1 min, and a final extension of 72°C for 10 min. A 10 μl of amplified product was run in a 1% agarose gel stained with ethidium bromide. Amplified products were visualized and photographed under UV light. PCR analysis confirmed identification of Neisseria meningitidis Group C. These results allowed us to confirm that the womans death was due to fulminant sepsis from group C N. meningitidis, as suggested by the clinical and premortem microbiological exams. More remarkably, these findings pointed out that the pathogen was still alive in various organs of the corpse at 11 days after death and able to grow in culture. The other case concerns a 6-month-old girl who was brought to the Emergency Department with a high fever (up to 39°C). A few hours later, the baby presented with cyanosis and breathing difficulty, and then she suddenly died. A forensic autopsy was performed at 7 days after death. The macroscopic examination revealed pulmonary edema and bilateral adrenal hemorrhage. During the autopsy, namely before internal examination, blood (from neck vessels), and cerebrospinal fluid (through lumbar puncture) samples were collected with sterile techniques for subsequent microbiological examinations. The methods here previously mentioned were used. N. meningitidis was found to grow in cultures of blood and cerebrospinal fluid samples after incubation for 48 h, thus providing the postmortem diagnosis of sepsis due to N. meningitidis. A premortem diagnosis was not possible because the fulminant disease caused the baby to die very quickly. Indeed, in this second case, the detection of N. meningitidis in postmortem cultures was crucial for the determination of the cause of the death. However, the most remarkable finding was the detection of N. meningitidis in cultures of samples preserved for 7 days after the death of the patient. To the best of our knowledge, there is only one other case of late postmortem detection of N. meningitidis reported in the literature. In this 2013 case report, group B N. meningitidis was identified in a putrefied corpse of a man who was found dead at home (Maujean et al., 2013). The last time that this man had been seen alive was about 10 days previously. No other information regarding the time of death was available. Formerly, the persistence of N. meningitidis in corpses has been identified only after a few hours after death, between 4 and 10 h (Ploy et al., 2005). In contrast, the observations presented here suggest that N. meningitidis can survive for more than 10 days after the death of an infected subject. The factors that can contribute to the growth of the bacteria in corpses are various. The abundance of nutritional elements like iron, amino acids, and other carbon sources is likely to be of importance, in combination with the absence of host defenses in corpses (Zughaier et al., 2014). However, these findings indicate that pathologists as well as other mortuary workers should exercise special precautions when working with corpses infected with N. meningitidis because the biological fluids and tissues of the corpses are still infectious for many days after death. We believe that these two reports are remarkable because they improve the current knowledge regarding infections due to N. meningitidis, an extremely relevant pathogen that causes a high mortality rate, especially among young people.