Metabolomics investigation of post-mortem human pericardial fluid.

INTRODUCTION: Due to its peculiar anatomy and physiology, the pericardial fluid is a biological matrix of particular interest in the forensic field. Despite this, the available literature has mainly focused on post-mortem biochemistry and forensic toxicology, while to the best of authors' knowledge post-mortem metabolomics has never been applied. Similarly, estimation of the time since death or post-mortem interval based on pericardial fluids has still rarely been attempted. OBJECTIVES: We applied a metabolomic approach based on 1H nuclear magnetic resonance spectroscopy to ascertain the feasibility of monitoring post-mortem metabolite changes on human pericardial fluids with the aim of building a multivariate regression model for post-mortem interval estimation. METHODS: Pericardial fluid samples were collected in 24 consecutive judicial autopsies, in a time frame ranging from 16 to 170 h after death. The only exclusion criterion was the quantitative and/or qualitative alteration of the sample. Two different extraction protocols were applied for low molecular weight metabolites selection, namely ultrafiltration and liquid-liquid extraction. Our metabolomic approach was based on the use of 1H nuclear magnetic resonance and multivariate statistical data analysis. RESULTS: The pericardial fluid samples treated with the two experimental protocols did not show significant differences in the distribution of the metabolites detected. A post-mortem interval estimation model based on 18 pericardial fluid samples was validated with an independent set of 6 samples, giving a prediction error of 33-34 h depending on the experimental protocol used. By narrowing the window to post-mortem intervals below 100 h, the prediction power of the model was significantly improved with an error of 13-15 h depending on the extraction protocol. Choline, glycine, ethanolamine, and hypoxanthine were the most relevant metabolites in the prediction model. CONCLUSION: The present study, although preliminary, shows that PF samples collected from a real forensic scenario represent a biofluid of interest for post-mortem metabolomics, with particular regard to the estimation of the time since death.

PMI estimation through metabolomics and potassium analysis on animal vitreous humour.

INTRODUCTION: The estimation of post-mortem interval (PMI) remains a major challenge in forensic science. Most of the proposed approaches lack the reliability required to meet the rigorous forensic standards. OBJECTIVES: We applied 1H NMR metabolomics to estimate PMI on ovine vitreous humour comparing the results with the actual scientific gold standard, namely vitreous potassium concentrations. METHODS: Vitreous humour samples were collected in a time frame ranging from 6 to 86 h after death. Experiments were performed by using 1H NMR metabolomics and ion capillary analysis. Data were submitted to multivariate statistical data analysis. RESULTS: A multivariate calibration model was built to estimate PMI based on 47 vitreous humour samples. The model was validated with an independent test set of 24 samples, obtaining a prediction error on the entire range of 6.9 h for PMI < 24 h, 7.4 h for PMI between 24 and 48 h, and 10.3 h for PMI > 48 h. Time-related modifications of the 1H NMR vitreous metabolomic profile could predict PMI better than potassium up to 48 h after death, whilst a combination of the two is better than the single approach for higher PMI estimation. CONCLUSION: The present study, although in a proof-of-concept animal model, shows that vitreous metabolomics can be a powerful tool to predict PMI providing a more accurate estimation compared to the widely studied approach based on vitreous potassium concentrations.

A Fatal Case of Metastatic Pulmonary Calcification during the Puerperium.

We present an unusual case of a fatal respiratory failure in a young woman developed two weeks after she gave birth at home. Circumstantial and clinical features of the case were strongly suggestive for a 'classical' septic origin of the respiratory symptoms. Autopsy, together with histopathological and immunohistochemical analyses allowed demonstrating a massive calcium redistribution consisting of an important osteolysis, especially from cranial bones and abnormal accumulation in lungs and other organs. Such physiopathology was driven by a primary hyperparathyroidism secondary to a parathyroid carcinoma as demonstrated by immunohistochemistry. This very rare case is furthermore characterised by a regular pregnancy course, ended with the birth of a healthy new-born. A complex interaction between pregnancy physiology and hyperparathyroidism might be hypothesised, determining the discrepancy between the relative long period of wellness and the tumultuous cascade occurred in the puerperium.

The Influence of Eyelid Position and Environmental Conditions on the Corneal Changes in Early Postmortem Interval: A Prospective, Multicentric OCT Study.

In the current study, using portable optical coherence tomography, we evaluated 46 corneas of 23 individuals in a multicenter setting during the first 17 h after death. Twenty-three eyes were kept open, and twenty three were kept closed. Furthermore, the experiment was carried out for 12 samples in summer and 11 in winter. Our data show that postmortem corneal alterations largely depend on the phenomena of dehydration (in particular in open eyes) and swelling of the stroma in closed eyes, probably due in the first phase to hypoxia/anoxia and subsequently to the passage by osmosis of the aqueous humor from the anterior chamber to the corneal tissue. Our findings could have significant repercussions in forensic pathology for estimating the postmortem interval and transplantation to optimize the conservation of the tissue before the explant.

Dual Corneal-Graft Rejection after mRNA Vaccine (BNT162b2) for COVID-19 during the First Six Months of Follow-Up: Case Report, State of the Art and Ethical Concerns.

Present mass vaccination against Coronavirus Disease-19 (COVID-19) is the most widely used health policy and the most promising approach to curb the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic globally. However, new side effects are emerging from the mass vaccination not described during the experimental stages. In the present study, we discuss a case of acute corneal graft rejection, which has occurred 25 years after transplantation and 13 days after the administration of the BNT162b2 vaccine (Comirnaty, BioNTech/Pfizer), which was followed-up for a period of six months. In this period, the corneal inflammation appeared twice but was successfully managed with topical therapy and supplementation of Vitamin D. A risk of corneal graft rejection must be included in the list of potential vaccine complications, in order to inform the transplanted patient to undergo a preliminary and a follow-up ocular examination, and eventually to include corneal graft in the list of contraindications to vaccination.

Looking for Post-Mortem Metabolomic Standardization: Waiting for Godot-The Importance of Post-Mortem Interval in Forensic Metabolomics.

A growing body of evidence suggests that the post-mortem interval exerts a strong effect on the metabolome, independently of the biological matrix or the cause of death. A sound and shared approach in standardization is mandatory.

The "Quarantine Dry Eye": The Lockdown for Coronavirus Disease 2019 and Its Implications for Ocular Surface Health.

The pandemic of coronavirus disease 2019 (COVID-19) has led many countries of the world to impose a series of containment measures such as lockdowns (mass quarantines), curfews or similar restrictions (eg, stay-at-home orders, or shelter-in-place orders). All these restrictions were established in order to limit spread of COVID-19. Thus, approximately 3.9 billion people worldwide were under lockdown by early April 2020. During this time (home confinement), some solutions have been proposed by experts to improve work and school productivity, including smart working and online school lessons. However, many of the restrictive measures are likely to act as predisposing factors for dry eye disease (DED), directly or related to sick building syndrome (SBS). Herein, we discuss the implications of quarantine measures on eye health, in particular on DED associated with SBS, and introduce some potential preventive strategies for lockdown-related ocular surface disorders. Several risk factors are implicated in their pathogenesis, including environmental changes (eg, air quality) and modifications in personal behaviors (eg, the abuse of digital devices, malnutrition, and sleep/psychiatric disorders). Considering a number of predisposing factors for DED, it is possible to state that patients under lockdown are at risk of ocular surface alterations. Accordingly, the COVID-19 pandemic era is expected to determine an increase in dry eye patients all around the world (a new phenomenon that we propose to name the "quarantine dry eye") in the event that the restrictive measures will be recursively extended over time.

Postmortem Ocular Findings in the Optical Coherence Tomography Era: A Proof of Concept Study Based on Six Forensic Cases.

Postmortem analysis of the ocular globe is an important topic for forensic pathology and transplantology. Although crucial elements may be gathered from examining cadaveric eyes, the latter do not routinely undergo in-depth analysis. The paucity of quantitative and objective data that are obtainable using current, invasive necroscopic techniques is the main reason for the limited interest in this highly specialized procedure. The aim of the current study is to describe and to object for the first time the postmortem ocular changes by mean of portable optical coherence tomography for evaluating ocular tissues postmortem. The design involved the postmortem analysis (in situ, and without enucleation) of 12 eyes by portable spectral-domain Optical Coherence Tomography. The scans were performed, in corneal, retinal and angle modality at different intervals: <6 h, 6th, 12th, and 24th hour and after autopsy (25th-72nd hour). The morphological changes in the cornea, sclera, vitreous humor and aqueous humor were easy to explore and objectify in these tissues in first 72 h postmortem. On the other hand, the "in situ" observation of the retina was difficult due to the opacification of the lenses in the first 24 h after death.

Comparative use of aqueous humour 1H NMR metabolomics and potassium concentration for PMI estimation in an animal model.

Estimation of the post-mortem interval (PMI) remains a matter of concern in the forensic scenario. Traditional and novel approaches are not yet able to fully address this issue, which relies on complex biological phenomena triggered by death. For this purpose, eye compartments may be chosen for experimental studies because they are more resistant to post-mortem modifications. Vitreous humour, in particular, has been extensively investigated, with potassium concentration ([K+]) being the marker that is better correlated with PMI estimation. Recently, a 1H nuclear magnetic resonance (NMR) metabolomic approach based on aqueous humour (AH) from an animal model was proposed for PMI estimation, resulting in a robust and validated regression model. Here we studied the variation in [K+] in the same experimental setup. [K+] was determined through capillary ion analysis (CIA) and a regression analysis was performed. Moreover, it was investigated whether the PMI information related to potassium could improve the metabolome predictive power in estimating the PMI. Interestingly, we found that a part of the metabolomic profile is able to explain most of the information carried by potassium, suggesting that the rise in both potassium and metabolite concentrations relies on a similar biological mechanism. In the first 24-h PMI window, the AH metabolomic profile shows greater predictive power than [K+] behaviour, suggesting its potential use as an additional tool for estimating the time since death.

The architecture of corneal stromal striae on optical coherence tomography and histology in an animal model and in humans.

The purpose of this study was to use a portable optical coherence tomography (OCT) for characterization of corneal stromal striae (CSS) in an ovine animal model and human corneas with histological correlation, in order to evaluate their architectural pattern by image analysis. Forty-six eyes from female adult sheep (older than 2 years), and 12 human corneas, were included in our study. The eyes were examined in situ by a portable OCT, without enucleation. All OCT scans were performed immediately after death, and then the eyes were delivered to a qualified histology laboratory. In the ovine animal model, CSS were detected with OCT in 89.1% (41/46) of individual scans and in 93.4% (43/46) of histological slices. In human corneas, CSS were found in 58.3% (7/12) of cases. In both corneal types, CSS appeared as "V"- or "X"-shaped structures, with very similar angle values of 70.8° ± 4° on OCT images and 71° ± 4° on histological slices (p ≤ 0.01). Data analysis demonstrated an excellent degree of reproducibility and inter-rater reliability of measurements (p < 0.001). The present study demonstrated that by using a portable OCT device, CSS can be visualized in ovine and human corneas. This finding suggests their generalized presence in various mammals. The frequent observation, close to 60%, of such collagen texture in the corneal stroma, similar to a 'truss bridge' design, permits to presume that it plays an important structural role, aimed to distribute tensile and compressive forces in various directions, conferring resilience properties to the cornea.

Repeatability and reproducibility of post-mortem central corneal thickness measurements using a portable optical coherence tomography system in humans: a prospective multicenter study.

To assess the repeatability and reproducibility of post-mortem central corneal thickness (CCT) measurements made by the portable iVue spectra-domain (SD) optical coherence tomography OCT (Optovue Inc, Fremont, CA) system in humans, and to prospectively establish the time-course of CCT after death. In a prospective multicenter setting, CCT measurements were obtained from 58 human eyes at the following 16 time-points after death: immediately (within 2 h), and at each hour by the next 17 h. The range of CCT values for each subject was determined and longitudinal data were used to illustrate the variation in open and close eye mode. All measurements were made by two independent and well-trained examiners for session. Main outcome measures were intraclass correlation coefficients (ICC), repeatability and reproducibility coefficients, and coefficients of variation of the average central (0-2 mm). Overall, a total of 5,568 OCT measurements were performed by examiners. The repeatability coefficient varied from 0.3 to 1.7% and the reproducibility coefficient varied from 0.3 to 1.6% throughout the entire experimental time frame. Furthermore, the values of the different ICCs were also high during the different postmortem intervals, thus demonstrating the excellent repeatability and reproducibility of the present OCT approach. When CCT measurements were analyzed longitudinally, corneal thickness showed different behavior based on the open or close eye mode. The present study demonstrates that portable OCT imaging can be reliably used for corneal pachymetric measurements in supine subjects and during the post mortem period, i.e. without visual fixation and normal physiology/architecture of examined tissues.

Safety Recommendations and Medical Liability in Ocular Surgery during the COVID-19 Pandemic: An Unsolved Dilemma.

Ocular surgery is one of the most performed medical procedures in the world. Its limitation or suspension, recursively extended over time, could be associated with a significant increase in the number of blind people worldwide. Indeed, cataract causes more than half of all cases of visual impairment in those countries with limited availability of means for performing eye operations (e.g., Africa or India). In this scenario, the pandemic of coronavirus disease 2019 (COVID-19) quickly resulted in the suspension or sharp reduction of various ophthalmic activities considered non-urgent, including lens replacement surgery or some intraocular injections. Despite the imperative need to continuously practice eye operations to avoid the abovementioned problems, there are currently little-shared and vague recommendations among the various countries on safety in operating rooms (for health care workers and patients) and poor legal protection for surgeons (potentially transmitting the COVID-19 infectious agent). Herein, we individuated and discussed some critical points in safety recommendations and medical liability. A paradigm shift for ocular surgery during the COVID-19 era is now mandatory. While telemedicine has been able to solve some problems in clinical ophthalmology, the lack of adequate health and legal protection for surgeons and patients may result in an excessive reduction in the volume of surgical interventions during a pandemic era and the immediately following period, thus determining inability to ensure health care to all patients.

The Ocular Surface and the Coronavirus Disease 2019: Does a Dual 'Ocular Route' Exist?

Coronavirus disease 2019 (COVID-19) is an important health problem that was defined as a pandemic by the World Health Organization on 11 March 2020. Although great concern has been expressed about COVID-19 infection acquired through ocular transmission, its underlying mechanism has not currently been clarified. In the current work, we analyzed and elucidated the two main elements that should be taken into account to understand the "ocular route", both from a clinical and molecular point of view. They are represented by the dynamism of the ocular surface system (e.g., the tear film turnover) and the distribution of ACE2 receptors and TMPRSS2 protein. Although it seems, at the moment, that there is a low risk of coronavirus spreading through tears, it may survive for a long time or replicate in the conjunctiva, even in absence of conjunctivitis signs, indicating that eye protection (e.g., protective goggles alone or in association with face shield) is advisable to prevent contamination from external droplets and aerosol.

Global Spread of Coronavirus Disease 2019 and Malaria: An Epidemiological Paradox in the Early Stage of A Pandemic.

In the current work, we discovered and analyzed the epidemiological paradox between coronavirus disease 2019 (COVID-19) and malaria in the initial phase of the ongoing pandemic. From the analysis of distribution data, the endemic presence of malaria seems to protect some populations from COVID-19 outbreak, particularly in the least developed countries. In this sense, molecular and genetic variations associated with malaria (e.g., in ACE2) might play a protective role against coronavirus infection. Moreover, the mechanism of action of some antimalarial drugs, e.g., the antiviral function, suggests their potential role in the chemoprophylaxis of coronavirus epidemics, despite possible adverse effects (e.g., retinal toxicity). All these data provide important insights to understand the spreading mechanisms of COVID-19, and to direct scientific research toward the study of some currently available medications.

Fourier-Domain OCT Imaging of the Ocular Surface and Tear Film Dynamics: A Review of the State of the Art and an Integrative Model of the Tear Behavior During the Inter-Blink Period and Visual Fixation.

In the last few decades, the ocular surface and the tear film have been noninvasively investigated in vivo, in a three-dimensional, high resolution, and real-time mode, by optical coherence tomography (OCT). Recently, OCT technology has made great strides in improving the acquisition speed and image resolution, thus increasing its impact in daily clinical practice and in the research setting. All these results have been achieved because of a transition from traditional time-domain (TD) to Fourier-domain (FD) technology. FD-OCT devices include a spectrometer in the receiver that analyzes the spectrum of reflected light on the retina or ocular surface and transforms it into information about the depth of the structures according to the Fourier principle. In this review, we summarize and provide the state-of-the-art in FD-OCT imaging of the ocular surface system, addressing specific aspects such as tear film dynamics and epithelial changes under physiologic and pathologic conditions. A theory on the dynamic nature of the tear film has been developed to explain the variations within the individual compartments. Moreover, an integrative model of tear film behavior during the inter-blink period and visual fixation is proposed.

COVID-19 and Italian Healthcare Workers From the Initial Sacrifice to the mRNA Vaccine: Pandemic Chrono-History, Epidemiological Data, Ethical Dilemmas, and Future Challenges.

On March 11, 2020, the World Health Organization (WHO) declared the coronavirus disease 2019 (COVID-19) outbreak a pandemic. Simultaneously, in Italy, in which the first case had occurred on February 18, the rigid phase of the lockdown began. The country has attracted worldwide attention, becoming at the same time a field of study both concerning the spread of the pandemic and advanced assessments of the effectiveness of political, public health, and therapeutic measures. The protagonists of the Italian crisis were the healthcare workers (HCWs) who were exposed to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) without having any perception of what they were facing, courageously contributing to the containment of the epidemic to be defined by the media as "heroes." However, in the first phase of the pandemic (March-May 2020), the price that the Italian Public Health System had to pay both in terms of the number of positive virus cases and deaths among the HCWs was beyond and represented a peculiarity compared to what happened in other countries. In the current study, after a summary of the evolution of the pandemic in Italy, we offer an analysis of the statistical data concerning contagions and deaths among healthcare workers (physicians in particular). In conclusion, we describe the critical issues that still need to be resolved and the future challenges facing healthcare workers and the general population.

Optical coherence tomography in forensic sciences: a review of the literature.

Optical coherence tomography (OCT) is an interferometric imaging technique that has revolutionized clinical ophthalmology since the first half of the 1990's. Despite this approach being successfully employed in ophthalmology and having great potential in forensic cases, its use in different forensic fields appears to be quite limited. In this review we reviewed the scientific literature regarding the application of OCT in forensic science and legal medicine from 1995 to 2019. Our research showed the usefulness of this approach for the study of coronary injuries, postmortem ocular changes, forensic entomology, and several other applications of specific forensic interest (the study of blood stains, fingerprints, and hair bulbs for personal identification, as well as the study of materials found in the crime scene for comparation, or anti-fraud investigation). The creation of specific 'ad hoc' devices and a better knowledge of this type of technology by pathologists will be a fundamental step to continue to develop the use of OCT forensic fields.