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Background: Pericardial fluid (PF) contains cells, proteins, and inflammatory mediators, such as cytokines, chemokines, growth factors, and matrix metalloproteinases. To date, we lack an adequate understanding of the inflammatory response that acute injury elicits in the pericardial space., Objective: To characterize the inflammatory profile in the pericardial space acutely after ischemia/reperfusion., Methods: Pigs were used to establish a percutaneous ischemia/reperfusion injury model. PF was removed from pigs at different time points postanesthesia or postischemia/reperfusion. Flow cytometry was used to characterize the immune cell composition of PF, while multiplex analysis was performed on the acellular portion of PF to determine the concentration of inflammatory mediators. There was a minimum of 3 pigs per group., Results: While native PF mainly comprises macrophages, we show that neutrophils are the predominant inflammatory cell type in the pericardial space after injury. The combination of acute ischemia/reperfusion (IR) and repeatedly accessing the pericardial space significantly increases the concentration of interleukin-1 beta (IL-1β) and interleukin-1 receptor antagonist (IL-1ra). IR significantly increases the pericardial concentration of TGFβ1 but not TGFβ2. We observed that repeated manipulation of the pericardial space can also drive a robust pro-inflammatory response, resulting in a significant increase in immune cells and the accumulation of potent inflammatory mediators in the pericardial space., Conclusion: In the present study, we show that both IR and surgical manipulation can drive robust inflammatory processes in the pericardial space, consisting of an increase in inflammatory cytokines and alteration in the number and composition of immune cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Heart failure (HF) is still the main cause of mortality worldwide. This study investigated the characteristics of human pericardial fluid-derived cells (hPFCs) and their effects in treating doxorubicin (DOX)-induced HF rats through intrapericardial injection. hPFCs were isolated from patients who underwent heart transplantation ( N = 5). These cells that primarily expressed SCA-1, NANOG, and mesenchymal markers, CD90, CD105, and CD73, were able to form adipocytes, osteoblasts, and cardiomyocytes in vitro. Passage 3 hPFCs (2.5 × 10 5 cells/heart) were injected into the pericardial cavity of the DOX-injured rat hearts, significantly improving cardiac functions after 4 weeks. The tracked and engrafted red fluorescent protein-tagged hPFCs coexpressed cardiac troponin T and connexin 43 after 4 weeks in the host myocardium. This observation was also coupled with a significant reduction in cardiac fibrosis following hPFC treatment (P < 0.0001 vs. untreated). The elevated inflammatory cytokines interleukin (IL)-6, IL-10, and tumor necrosis factor-α in the DOX-treated hearts were found to be significantly reduced ( P < 0.001 vs. untreated), while the regional proangiogenic vascular endothelial growth factor A (VEGFA) level was increased in the hPFC-treated group after 4 weeks ( P < 0.05 vs. untreated). hPFCs possess stem cell characteristics and can improve the cardiac functions of DOX-induced HF rats after 4 weeks through pericardial administration. The improvements were attributed to a significant reduction in cardiac fibrosis, inflammation, and elevated regional proangiogenesis factor VEGFA, with evidence of cellular engraftment and differentiation in the host myocardium.

The potential of the pericardial space as a therapeutic delivery tool for cardiac fibrosis and heart failure (HF) treatment has yet to be elucidated. Recently, miRNAs and exosomes have been discovered to be present in human pericardial fluid (PF). Novel studies have shown characteristic human PF miRNA compositions associated with cardiac diseases and higher miRNA expressions in PF compared to peripheral blood. Five key studies found differentially expressed miRNAs in HF, angina pectoris, aortic stenosis, ventricular tachycardia, and congenital heart diseases with either atrial fibrillation or sinus rhythm. As miRNA-based therapeutics for cardiac fibrosis and HF showed promising results in several in vivo studies for multiple miRNAs, we hypothesize a potential role of miRNA-based therapeutics delivered through the pericardial cavity. This is underlined by the favorable results of the first phase 1b clinical trial in this emerging field. Presenting the first human miRNA antisense drug trial, inhibition of miR-132 by intravenous administration of a novel antisense oligonucleotide, CDR132L, established efficacy in reducing miR-132 in plasma samples in a dose-dependent manner. We screened the literature, provided an overview of the miRNAs and exosomes present in PF, and drew a connection to those miRNAs previously elucidated in cardiac fibrosis and HF. Further, we speculate about clinical implications and potential delivery methods., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Exosomes are nanovesicles 30-150 nm in diameter released extracellularly. Those isolated from human body fluids reflect the characteristics of their cells or tissues of origin. Exosomes carry extensive biological information from their parent cells and have significant potential as biomarkers for disease diagnosis and prognosis. However, there are limited studies utilizing exosomes in postmortem diagnostics. In this study, we extended our initial research which identified the presence and established detection methodologies for exosomes in postmortem fluids. We analyzed exosomal miRNA extracted from plasma and pericardial fluid samples of a control group (n = 13) and subjects with acute myocardial infarction (AMI; n = 24). We employed next-generation sequencing (NGS) to investigate whether this miRNA could serve as biomarkers for coronary atherosclerosis leading to acute myocardial infarction. Our analysis revealed 29 miRNAs that were differentially expressed in the AMI group compared to the control group. Among these, five miRNAs exhibited more than a twofold increase in expression across all samples from the AMI group. Specifically, miR-486-5p levels were significantly elevated in patients with high-grade (type VI or above) atherosclerotic plaques, as per the American Heart Association criteria, highlighting its potential as a predictive biomarker for coronary atherosclerosis progression. Our results indicate that postmortem-derived exosomal microRNAs can serve as potential biomarkers for various human diseases, including cardiovascular disorders. This finding has profound implications for forensic diagnostics, a field critically lacking diagnostic markers.

Data-dependent liquid chromatography tandem mass spectrometry is challenged by the large concentration range of proteins in plasma and related fluids. We adapted the SCoPE method from single-cell proteomics to pericardial fluid, where a myocardial tissue carrier was used to aid protein quantification. The carrier proteome and patient samples were labeled with distinct isobaric labels, which allowed separate quantification. Undepleted pericardial fluid from patients with type 2 diabetes mellitus and/or heart failure undergoing heart surgery was analyzed with either a traditional liquid chromatography tandem mass spectrometry method or with the carrier proteome. In total, 1398 proteins were quantified with a carrier, compared to 265 without, and a higher proportion of these proteins were of myocardial origin. The number of differentially expressed proteins also increased nearly four-fold. For patients with both heart failure and type 2 diabetes mellitus, pathway analysis of upregulated proteins demonstrated the enrichment of immune activation, blood coagulation, and stress pathways. Overall, our work demonstrates the applicability of a carrier for enhanced protein quantification in challenging biological matrices such as pericardial fluid, with potential applications for biomarker discovery. Mass spectrometry data are available via ProteomeXchange with identifier PXD053450., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Risperidone (Ris) is a second-generation antipsychotic that belongs to the chemical class of benzisoxazole derivatives. 9-Hydroxy (9OH-) Ris is well known among the six reported metabolites of Ris and had been examined using not only blood but also other matrices, but the other five metabolites reported such as benzisoxazole ring-cleaved Ris (c-Ris) and c-9OH-Ris had been detected only in blood, urine and feces. In the present work, large peaks of c-Ris and c-9OH-Ris were detected in the liver, kidney, cerebrum, blood, pericardial fluid, bile and urine obtained from two cadavers. There is a potential that c-Ris and c-9OH-Ris will be good markers to prove Ris consumption in forensic toxicology cases. For example, the peak ratios of c-Ris against the parent Ris in the kidney and blood were as high as 3.9 and 3.6 in cadaver 1; and 7.0 and 7.9 in cadaver 2, respectively. In addition to the previously reported six metabolites, five new metabolites such as dehydrogenated-Ris, 7-keto-Ris and three benzisoxazole ring-cleaved metabolites were disclosed in the present work, and the pathways for the totally eleven metabolites detected in human solid tissues and body fluids have also been proposed, because such pathways were neither reported nor discussed previously., Competing Interests: Declaration of Competing Interest The authors declare that there are no financial or other relationships that could lead to a conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Pericardial fluid (PF) has been suggested as a reservoir of molecular targets that can be modulated for efficient repair after myocardial infarction (MI). Here, we set out to address the content of this biofluid after MI, namely in terms of microRNAs (miRs) that are important modulators of the cardiac pathological response. PF was collected during coronary artery bypass grafting (CABG) from two MI cohorts, patients with non-ST-segment elevation MI (NSTEMI) and patients with ST-segment elevation MI (STEMI), and a control group composed of patients with stable angina and without previous history of MI. The PF miR content was analyzed by small RNA sequencing, and its biological effect was assessed on human cardiac fibroblasts. PF accumulates fibrotic and inflammatory molecules in STEMI patients, namely causing the soluble suppression of tumorigenicity 2 (ST-2), which inversely correlates with the left ventricle ejection fraction. Although the PF of the three patient groups induce similar levels of fibroblast-to-myofibroblast activation in vitro, RNA sequencing revealed that PF from STEMI patients is particularly enriched not only in pro-fibrotic miRs but also anti-fibrotic miRs. Among those, miR-22-3p was herein found to inhibit TGF-β-induced human cardiac fibroblast activation in vitro. PF constitutes an attractive source for screening diagnostic/prognostic miRs and for unveiling novel therapeutic targets in cardiac fibrosis.

Soluble growth stimulation expressed gene 2 protein (sST2) is a myocardial protein induced by biomechanical stress. sST2 is widely present in the serum of patients with heart failure and is recommended as an important indicator to predict adverse outcomes in these patients. However, no postmortem biochemical analysis of sST2 in forensic practice has been reported. The present pilot study aimed to investigate the expression of sST2 in the pericardial fluid of patients with sudden cardiac death (SCD) caused by ischemic heart disease (IHD). In addition, to explore the relationship of sST2 with CK-MB, cTnT, and NT-proBNP, which have been proven to be auxiliary biomarkers for the diagnosis of SCD, we analyzed CK-MB, cTnT, NT-proBNP, and sST2 levels in twenty-one pericardial fluid samples from the Center of Forensic Investigation, China Medical University, with a Roche cobas e 411 electrochemiluminescence automatic immunoassay system and ST2/IL-33R Valukine™ enzyme-linked immunosorbent assay kit. The levels of sST2 in the pericardial fluid of patients with SCD caused by IHD were significantly increased (P < 0.01) and positively correlated with CK-MB and NT-proBNP (P < 0.0001). Receiver operating characteristic curve analysis indicated that the combined measurement of sST2 and NT-proBNP has a higher diagnostic value for SCD caused by IHD than the measurement of either indicator alone. This study preliminarily demonstrated that sST2 in the pericardial fluid was significantly increased in patients with SCD caused by IHD and might be used as a novel auxiliary biomarker for postmortem diagnosis of SCD in forensic practice., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background and Objective: Pericardial Fluid (PF) is a rich reservoir of biologically active factors. Due to its proximity to the heart, the biochemical structure of PF may reflect the pathological changes in the cardiac interstitial environment. This manuscript aimed to determine whether the PF level of cardiac troponins changes in patients undergoing cardiac surgery., Methods: This scoping review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Medline, EMBASE, Cochrane, ClinicalTrials.gov, and Google Scholar databases were electronically searched for primary studies using the keywords "pericardial fluid," "troponin," and "cardiac surgery." The primary outcome of interest was changes in troponin levels within the PF preoperatively and postoperatively. Secondary outcomes of interest included comparisons between troponin level changes in the PF compared to plasma., Results: A total of 2901 manuscripts were screened through a title and abstract stage by two independent blinded reviewers. Of those, 2894 studies were excluded, and the remaining seven studies underwent a full-text review. Studies were excluded if they did not provide data or failed to meet inclusion criteria. Ultimately, six articles were included that discussed cardiac troponin levels within the PF in patients who had undergone cardiac surgery. Pericardial troponin concentration increased over time after surgery, and levels were significantly higher in PF compared to serum. All studies found that the type of operation did not affect these overall observations., Conclusion: Our review of the literature suggest that the PF level of cardiac troponins increases in patients undergoing cardiac surgery, irrespective of the procedure type. However, these changes' exact pattern and clinical significance remain undefined., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Post-operative atrial fibrillation (POAF) is a common complication of cardiac surgery which is associated with longer hospital stay, diminished quality of life, and increased mortality. Yet, the pathophysiology of POAF is poorly understood and it is unclear which patients are at highest risk. Pericardial fluid (PCF) analysis is emerging as an important tool for the early detection of biochemical and molecular changes in the cardiac tissue. With the epicardium acting as a semi-permeable membrane, the composition of PCF reflects the activity of the cardiac interstitium. Emerging research on PCF composition has identified promising biomarkers which may help stratify the risk for developing POAF. These include inflammatory molecules, such as interleukin-6, mitochondrial deoxyribonucleic acid, and myeloperoxidase, as well as natriuretic peptides. Additionally, PCF appears to be superior to serum analysis in detecting changes in these molecules during the early postoperative period after cardiac surgery. The aim of the present narrative review is to summarize the current literature on the temporal changes in the levels of potential biomarkers in PCF after cardiac surgery and their association with the development of new-onset postoperative atrial fibrillation., Competing Interests: Declaration of Competing Interest None declared by all authors., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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 1 H 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 1 H 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., (© 2023. The Author(s).)

Tissue engineering applications are widely used to repair and regenerate damaged tissues and organs. A scaffold, which is an important component in tissue engineering, provides a 3D environment for cells. In this study, the usability of PF components for the production of an ideal scaffold was investigated. For this aim, pericardial fluid (PF) was harvested from the bovine heart, then its structure and components were characterized. The results of Raman spectroscopy analysis, histological staining, and scanning electron microscopy (SEM) shows that the pericardial fluid contains collagen type I and IV, elastin, fibrin, and glycosaminoglycan (GAG), which are native extracellular matrix (ECM) components. The results demonstrated that (i) PF contains native ECM proteins and GAG such as collagen types I, III, and IV, elastin, and fibrin. (ii) The PF is highly similar to the native ECM structure. (iii) PF can significantly contribute to many tissue engineering studies as a native ECM material to increase the biocompatibility of biomaterials and to several in vitro/in vivo cell culture studies. (iv) PF containing multiple ECM molecules, can be used alone or together with hyaluronic acid, poly(ethylene glycol) (PEG), alginate, chitosan, matrigel, and gelatin methacryloyl (GelMA) materials in bioprinting systems for eliminating the disadvantages of these materials., (© 2023 Wiley Periodicals LLC.)

Lectin-like oxidized LDL receptor-1 (LOX-1) is the endothelial receptor for oxidized LDL. This receptor's extracellular domain is released into the blood as soluble LOX-1 (sLOX-1) and has been linked to ischemic heart disease (IHD), cerebrovascular diseases (CVDs), obesity, and diabetes. We recently reported that sLOX-1 fluid levels in postmortem pericardial fluid were comparable to clinical values in live patients and that significant increases in sLOX-1 were observed in patients with IHD. However, postmortem serum and urine sLOX-1 levels were higher than serum levels in living patients. Here, we conducted LOX-1 immunostaining in forensic specimens (aorta and heart) and evaluated pericardial fluid sLOX-1 in 221 medicolegal autopsy cases (67 IHD, 11 CVD, 17 inflammatory diseases, and 126 control cases) with a postmortem interval < 72 h to assess the diagnostic efficiency of postmortem pericardial fluid sLOX-1. Furthermore, we evaluated the relationships between pericardial fluid sLOX-1 and body mass index (BMI), blood HbA1c, serum C-reactive protein (CRP), high-density lipoprotein cholesterol (HDL-C), and low-density-lipoprotein cholesterol (LDL-C). LOX-1 immunostaining positivity was found in the aortic intima. Pericardial fluid sLOX-1 levels were considerably higher in patients with IHD and CVD. However, there were no significant differences in patients with inflammatory diseases and controls. No associations between pericardial fluid sLOX-1 and BMI, HbA1c, CRP, HDL-C, or LDL-C were found. These results indicate sLOX-1 utility in the postmortem diagnosis of IHD and CVD., Competing Interests: Declaration of Competing Interest Disclosure of potential conflicts of interest: Not applicable., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The purpose of this study was to investigate the regulatory mechanism of miR-450a-2-3p in myocardial fibrosis in patients with atrial fibrillation. For this purpose, the expression profile of GSE55296 was extracted from the GEO database, and differentially expressed lncRNAs were identified. Gene ontology analysis of the target genes of mir-450a-2-3p indicated that there was a regulatory relationship between LINC00636 and miR-450a-2-3p. Further, the expression levels of the analyzed RNAs were confirmed by RT-qPCR. TGF- β 1-induced cardiac fibroblasts (CFs) and human umbilical vein endothelial cells (HUVECs) were used to establish a myocardial fibrosis model and endothelium-mesenchymal transformation (EMT) model in vivo. We hypothesized that exosomes containing LINC00636 regulate the expression of miR-450a-2-3p. LINC00636 was positively correlated with the expression of miR-450a-2-3p. The overexpression of miR-450a-2-3p suppressed the MAPK1 expression in CFs, thereby inhibiting the expression of α -SMA, COL1, and COL3 and preventing CF proliferation. In HUVECs, the miR-450a-2-3p overexpression upregulated the expression of VE-Cadherin (VE-Cad) and platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) by inhibiting the mitogen-activated protein kinase 1 (MAPK1) expression, whereas the expression levels of vimentin, COL1, and COL3 decreased. These results indicate that LINC00636, which is present in human pericardial fluid, is an antifibrotic molecule that inhibits MAPK1 through the miR-450a-2-3p overexpression and improves cardiac fibrosis in patients with atrial fibrillation., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2021 Langsha Liu et al.)

Background: We evaluated malignancy according to the characteristics of pericardial fluid in symptomatic Japanese patients undergoing pericardiocentesis and computed tomography (CT)., Methods: This was a retrospective, single-center, observational study of 125 symptomatic patients undergoing pericardiocentesis. The patients were classified into two groups: a malignancy group and a non-malignancy group, according to the primary disease and cytology of the pericardial effusion (PE). We compared the pericardial fluid sample and CT measurements between both groups., Results: All patients were diagnosed as having exudative PE by Light's criteria. PE with malignant cells was demonstrated in 76.8% of the malignancy group patients. Pericardial to serum lactate dehydrogenase (LDH) ratio > 0.6, as one of Light's criteria, was associated with malignancy (p = 0.017). Lower serum brain natriuretic peptide (BNP) concentration was also associated with malignancy (BNP: 126.9 ± 89.8 pg/ml vs 409.2 ± 97.7 pg/ml, malignancy vs non-malignancy groups, respectively; p = 0.037). A significant difference was observed in pericardial fluid glucose level between the malignancy and non-malignancy groups (pericardial fluid glucose: 78.24 ± 48.29 mg/dl vs 98.41 ± 44.85, respectively; p = 0.048). Moreover, CT attenuation values (Hounsfield units (HU)) tended to be higher in the malignancy group vs the non-malignancy group (22.7 [interquartile range (IQR), 17.4-26.0] vs 17.4 [IQR, 13.7-26.4], respectively; p = 0.08). The sensitivity and specificity of pericardial fluid glucose level ≤ 70 mg/dl and CT attenuation values > 20 HU were 40.9% and 89.6%, respectively, in the malignancy group. The positive- and negative predictive values of pericardial fluid glucose level ≤ 70 mg/dl and CT attenuation values > 20 HU were 85.7% and 50.0%, respectively, in the malignancy group. Pericardial fluid glucose level ≤ 70 mg/dl and CT attenuation values > 20 HU were cutoff values associated with malignancy (p = 0.012)., Conclusions: Lower pericardial fluid glucose level with higher CT attenuation values may suggest malignancy-related PE.

Opioids are the drugs most commonly detected in overdose deaths and the second most consumed worldwide. An analytical methodology has been optimized and fully validated for the determination of codeine, morphine, 6-acetylmorphine, 6-acetylcodeine, oxycodone, oxymorphone and fentanyl in whole blood and pericardial fluid. The internal standards used were codeine-d3, morphine-d3, 6-acetylmorphine-d3 and fentanyl-d5. Before solid-phase extraction, volumes of 250 μL of blood and pericardial fluid were subjected to a protein precipitation (with 750 μL of ice-cold acetonitrile) and a microwave-induced oximation was performed using a solution of 1% aqueous hydroxylamine hydrochloride in phosphate-buffered saline (1:2, v/v). Finally, the dried extracts were further derivatized with a solution of n-methyl-n-(trimethylsilyl) trifluoroacetamide + 5% trimethylchlorosilane under microwave irradiation. The chromatographic analysis was carried out using gas chromatography-mass spectrometry operating in electron impact and selected ion monitoring mode. For all analytes, the method was linear between 5 and 1,000 ng/mL with determination coefficients (r2) >0.99. Depending on the analyte and matrix, the limit of detection varies between 3 and 4 ng/mL. Intra- and intermediate precision (<20%) and bias (±20%) were acceptable for all analytes in both matrices. The stability of the substances in the studied matrices was guaranteed, at least, 24 h in the autosampler, 4 h at room temperature and 30 days after three freeze/thaw cycles. This methodology was applied to real samples from the Laboratory of Chemistry and Forensic Toxicology, Centre Branch, of the National Institute of Legal Medicine and Forensic Sciences, Portugal., (© The Author(s) 2020. Published by Oxford University Press on behalf of The International Association of Forensic Toxicologists, Inc. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objective: Pregnancy-associated plasma protein-A (PAPP-A) has been suggested as a proatherogenic enzyme by its ability to locally increase insulin-like growth factor (IGF) activity through proteolytic cleavage of IGF binding protein-4 (IGFBP-4). Recently, stanniocalcin-2 (STC2) was discovered as an inhibitor of PAPP-A. This study aimed to investigate IGFBP-4, PAPP-A, and STC2 as local regulators of IGF bioactivity in the cardiac microenvironment by comparing levels in the pericardial fluid with those in the circulation of patients with cardiovascular disease., Methods: Plasma and pericardial fluid were obtained from 39 patients undergoing elective cardiothoracic surgery, hereof 15 patients with type 2 diabetes. Concentrations of IGF-I, intact and fragmented IGFBP-4, PAPP-A, and STC2 were determined by immunoassays and IGF bioactivity by a cell-based assay., Results: In pericardial fluid, the concentrations of total IGF-I, intact IGFBP-4, and STC2 were 72 ± 10%, 91 ± 5%, and 40 ± 24% lower than in plasma, while PAPP-A was 15 times more concentrated. The levels of the 2 IGFBP-4 fragments generated by PAPP-A and reflecting PAPP-A activity were elevated by more than 25%. IGF bioactivity was 62 ± 81% higher in the pericardial fluid than plasma. Moreover, pericardial fluid levels of both IGFBP-4 fragments correlated with the concentration of PAPP-A and with the bioactivity of IGF. All protein levels were similar in pericardial fluid from nondiabetic and diabetic subjects., Conclusions: PAPP-A increases IGF bioactivity by cleavage of IGFBP-4 in the pericardial cavity of cardiovascular disease patients. This study provides evidence for a distinct local activity of the IGF system, which may promote cardiac dysfunction and coronary atherosclerosis., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Biochemical markers undergo postmortem changes that complicate diagnostic measurement. C-reactive protein (CRP) is one marker that is known to be useful in postmortem specimens, with high levels reported in forensic cases of sepsis, trauma, and ketoacidosis. In the present study, we included 30 cases (17 males and 13 females) that underwent forensic autopsy within 80 h of death and had a CRP result from two postmortem specimens (serum from cardiac blood and pericardial fluid) and an emergency room specimen. Antemortem results were taken at a time near to cardiopulmonary arrest and the declaration of death. CRP levels in postmortem serum and pericardial fluid correlated with those in antemortem serum. Although no significant difference was observed between the antemortem and postmortem serum levels, the pericardial level was significantly low and five false negatives were observed. We conclude that postmortem serum is suitable for use in CRP measurement, and in cases with high antemortem CRP levels, postmortem pericardial fluid may be an appropriate alternative., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.)

Biochemical analysis of creatine kinase MB (CK-MB), which is a biomarker of myocardial damage, is used as a potential adjunct test in clinical and forensic medicine. However, there is no previous meta-analysis that summarizes the diagnostic value of postmortem biochemical analysis of CK-MB in cardiac death. The purpose of this study was to perform a systematic literature review and meta-analysis of postmortem CK-MB in cardiac death for forensic work. Six online databases, including PubMed, Embase, Cochrane Library, the China National Knowledge Infrastructure (CNKI), the China Biomedical Literature Database (CBM), and Wanfang Data, were used to search for related studies. The quality of the included literature was assessed according to the Newcastle-Ottawa Quality Assessment Scale (NOS). The meta-analysis was performed by Review Manager version 5.3 software to investigate the diagnostic role of postmortem CK-MB in cardiac death, especially in myocardial infarction. Sixteen pieces of related literature were identified, all of which were considered high quality. The results of the meta-analysis revealed that the postmortem CK-MB level in the pericardial fluid was significantly higher in the cardiac death group with a standard mean difference (SMD) = 0.63, 95% confidence interval (CI) = 0.09~1.17, p = 0.02. This was also the result in the myocardial infarction group (SMD = 0.83, 95% CI = 0.10~1.56, p = 0.03). No significant difference in CK-MB was found in serum for cardiac death (SMD = -0.31, 95% CI = -0.85~0.24, p = 0.27) or myocardial infarction (SMD = -0.10, 95% CI = -0.69~0.49, p = 0.74). The postmortem biochemical analysis of CK-MB in the pericardial fluid can be used as an auxiliary method in the postmortem diagnosis of cardiac death, along with autopsy and histological investigation.

Sudden cardiac death (SCD) is an unexpected death caused by a sudden loss of cardiac function, which is currently a global public health problem. Evaluation of the agonal cardiac function of the deceased is a quite important task for the diagnosis of SCD in forensic medicine. Brain natriuretic peptide (BNP) and N-terminal proBNP (NT-proBNP) are currently considered as significant biomarkers for the diagnosis of heart failure in both clinical and forensic practices. To investigate the postmortem evaluation roles of postmortem BNP and NT-proBNP levels for SCD, the present study meta-analyzed eight related studies from Embase, Cochrane Library, PubMed, China Biomedical Literature Database, China National Knowledge Infrastructure, and Wanfang Data. Newcastle-Ottawa Quality Assessment Scale was used to assess the quality of the included literature, and the meta-analysis was performed by RevMan 5.3.5 software. Postmortem NT-proBNP in pericardial fluid showed higher levels in the SCD group than that of the non-SCD group with the weighted mean difference = 3665.74, 95% confidence interval: 1812.89-5518.59, and p = 0.0001. However, postmortem levels of BNP in pericardial fluid and NT-proBNP in serum revealed no statistical difference between SCD and non-SCD subjects. The results of present meta-analysis demonstrated that postmortem NT-proBNP in the pericardial fluid could be used as an ancillary indicator for evaluation of agonal cardiac function in forensic medicine., (© 2019 American Academy of Forensic Sciences.)

Background: From the results of a previous study, it remained to be investigated if a perioperative rise of few tested coagulation and inflammation markers is caused by conventional cardiopulmonary bypass (CPB) itself or rather by direct recirculation of pericardial fluids., Methods: Forty-eight patients operated on with conventional CPB for myocardial revascularization were randomized either for direct recirculation of pericardial suction fluids or for cell saving (CS)., Results: Thrombin-antithrombin complexes showed lower values intraoperatively in the CS group ( p  < 0.0001), and D-dimers tended to remain lower at intensive care unit arrival ( p  = 0.095). Tests of inflammation markers were less meaningful., Conclusion: Direct recirculation of pericardial fluids rather than conventional CPB itself causes major intraoperative changes of some coagulation markers. Pericardial blood loss with direct recirculation should be kept to a minimum to avoid unnecessary activation of coagulation. Inflammation markers need further investigations., Competing Interests: The authors have no disclosure or conflict of interest., (Georg Thieme Verlag KG Stuttgart · New York.)

The utility of biochemical marker analysis in forensic autopsy cases is still uncertain due to the postmortem changes which they undergo. Thus, research is required to elucidate alternative samples and biochemical markers which are less affected by postmortem changes. Levels of N-terminal pro-brain natriuretic peptide (NT-proBNP) are known to be elevated in congestive heart failure (CHF), acute myocardial infarction (AMI), and sepsis patients. Although NT-proBNP is reportedly excreted into the urine, no study has previously evaluated the diagnostic efficacy of urinary concentrations in a forensic setting. The aim of this study was to evaluate the diagnostic efficacy of NT-proBNP concentration in urine obtained postmortem in a series of forensic autopsy cases., Methods: Urinary NT-proBNP was measured in 36 AMI, 10 CHF, and 19 sepsis cases, and in 124 control cases (all with postmortem interval [PMI]<72h)., Results: Urinary NT-proBNP was significantly higher in AMI, CHF, and sepsis cases than in control cases. Cut-off values for diagnosing AMI, CHF, and sepsis-related fatalities were 98 (sensitivity, 55.6 %; specificity, 73.4 %), 1050 (sensitivity, 80.0 %; specificity, 94.4 %), and 363pg/mL (sensitivity, 84.2 %; specificity, 85.5 %), respectively. Furthermore, we subdivided the control cases according to the death process as either acute death (87 cases) or prolonged death cases (37 cases). Although urine NT-proBNP of CHF and sepsis cases were significantly higher compared with both cases, the concentration in the AMI cases were significantly high only when compared with the acute death cases., Conclusion: This study is the first to elucidate the diagnostic utility of NT-proBNP measurement in urine obtained postmortem in a series of causes of death. This study suggests the diagnostic efficacy for AMI, CHF, and sepsis-related fatality in cases in which the PMI was within 72h., (Copyright © 2019 Elsevier B.V. All rights reserved.)

We reported the first comprehensive autopsy case of death due to intravenous injection of nicotine. We examined the distribution of nicotine in the body tissues and fluid and exposed the pathophysiology of nicotine poisoning. A 19-year-old woman was rushed to the hospital in cardiorespiratory arrest and was confirmed dead upon arrival. Liquid nicotine, hydrogen peroxide water, and a syringe were found in the hotel room where she stayed. On autopsy, nicotine concentration was the highest (15,023 μg/mg) in the tissue around the injection mark on the right upper arm. Among the body fluids, the intraperitoneal fluid had the highest, whereas the pericardial fluid had the lowest (0.736 μg/mL) nicotine concentration. Among the organs, the brain had the highest (11.637 μg/mg), whereas the fat tissue had the lowest (1.307 μg/mg) nicotine concentration. The concentration of cotinine, which is the metabolite of nicotine, was the highest in the tissue around the injection mark on the right arm (5.495 μg/mg) and was almost the same among the other body fluids and organs. The respective concentrations of nicotine and cotinine were 1.529 μg/mL and 0.019 μg/mL in the left heart blood and 3.157 μg/mL and 0.002 μg/mL in right heart blood. In this case, the nicotine concentrations in blood reached the lethal level. The distributions of nicotine and cotinine, as indicated by the intravenous injection, were related to the distribution of organs that metabolize nicotine and the distribution of nicotinic acetylcholine receptors.

Gene expression has become an interesting research area in forensic pathology to investigate the process of death at the molecular level. The aims of this study were to analyze changes in gene expression patterns in relation to the cause of death, and to propose new molecular markers of myocardial ischemia of potential use for the postmortem diagnosis of early ischemic heart damage in cases of sudden cardiac death (SCD). We determined mRNA levels of five proteins related with ischemic myocardial damage and repair - TNNI3, MYL3, TGFB1, MMP9 and VEGFA - in specific sites of the myocardium, blood and pericardial fluid in samples from 30 cadavers with different causes of death (SCD, multiple trauma, mechanical asphyxia, and other natural deaths). TNNI3 expression in blood, and MMP9 expression in pericardial fluid, were significantly higher when the cause of death was mechanical asphyxia, probably because of the more sensitive response of these proteins to acute systemic hypoxia/ischemia. Specifically, among SCD cases, increased MYL3, VEGFA and MMP9 values in the anterior wall of the right ventricle were found when the confirmed cause of death was acute myocardial infarction (AMI). Higher TGFB1 expression was found in the interventricular septum when AMI was not the cause of death, most likely as a reflection of the short duration of ischemia. Molecular biology techniques can provide complementary tools for the forensic diagnosis of early ischemic myocardial damage and AMI, and may make it possible to determine the duration and severity of myocardial ischemia., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: Intrapericardial fibrous adhesions increase the risk of sternal reentry. Proteoglycan 4/lubricin (PRG4) is a mucin-like glycoprotein that lubricates tissue compartments and prevents inflammation. We characterized PRG4 expression in human pericardium and examined its effects in vitro on human cardiac myofibroblast fibrotic activity and in vivo as a measure of its therapeutic potential to prevent adhesions., Methods: Full-length PRG4 expression was determined using Western blot analysis and amplified luminescent proximity homogeneous assay in human pericardial tissues obtained at cardiotomy. The in vitro effects of PRG4 were investigated on human cardiac myofibroblasts for cell adhesion, collagen gel contraction, and cell-mediated extracellular matrix remodeling. The influence of PRG4 on pericardial homeostasis was determined in a chronic porcine animal model., Results: PRG4 is expressed in human pericardial fluid and colocalized with pericardial mesothelial cells. Recombinant human PRG4 prevented human cardiac myofibroblast attachment and reduced myofibroblast activity assessed using collagen gel contraction assay (64.6% ± 8.1% vs 47.1% ± 6.8%; P = .02). Using a microgel assay, human cardiac myofibroblast mediated collagen fiber remodeling was attenuated by PRG4 (1.17 ± 0.03 vs 0.90 ± 0.05; P = .002). In vivo, removal of pericardial fluid alone induced severe intrapericardial adhesion formation, tissue thickening, and inflammatory fluid collections. Restoration of intrapericardial PRG4 was protective against fibrous adhesions and preserved the pericardial space., Conclusions: For the first time, we show that PRG4 is expressed in human pericardial fluid and regulates local fibrotic myofibroblast activity. Loss of PRG4-enriched pericardial fluid after cardiotomy might induce adhesion formation. Therapeutic restoration of intrapericardial PRG4 might prevent fibrous/inflammatory adhesions and reduce the risk of sternal reentry., (Copyright © 2018 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.)

Objectives: Alpha-fetoprotein (AFP) measurement in pericardial, peritoneal (ascites), and pleural fluids is sometimes requested by clinicians as supportive evidence in the evaluation of suspected malignancy. As commercially available, Food and Drug Administration (FDA)-cleared AFP assays are not validated for these fluid types, laboratories must complete additional validation studies to comply with regulatory requirements for body fluid testing. The objective of this study was therefore to conduct a matrix evaluation for these body fluid types using the Beckman Access AFP assay on the UniCel DxI 800 immunoassay system., Design and Methods: Using an Institutional Review Board (IRB) approved protocol, previously collected pericardial fluid, peritoneal fluid, pleural fluid, and serum specimens were de-identified and frozen at -20 °C prior to matrix evaluation experiments. Spiked recovery, mixed recovery/linearity, and precision studies were conducted., Results: In spiked and mixed recovery studies, the average percent (%) recovery was within predefined acceptable limits (±15%) for all three body fluids. Linearity was observed over the analytical measurement range (AMR) for all three body fluids (slope, intercept, systematic error): pericardial 0.988, -0.1, 6.1%; peritoneal 0.986, 0.0, 4.1%; and pleural 1.016, 0.0, 1.6%. Imprecision was ≤6.0% CV for all three body fluids at both high and low AFP concentrations., Conclusions: Matrix interference with AFP testing was not observed for pericardial, peritoneal, or pleural fluids on the Beckman UniCel DxI 800 system., (Copyright © 2018 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Purpose: Ischemic heart disease (IHD) is a major cause of death in developed countries. Postmortem IHD diagnosis using biochemical markers is difficult because of the postmortem changes. In the present study, we investigated the utility of soluble lectin-like low-density lipoprotein receptor-1 (sLOX-1) in body fluids obtained from forensic autopsy cases., Methods: We measured pericardial fluid, urine, and serum sLOX-1 levels; these samples were obtained from medicolegal autopsy cases (n = 149, postmortem interval <72 h), and the utility of these biomarkers postmortem acute IHD diagnosis was evaluated., Results: The pericardial fluid and urine of patients with acute IHD had higher sLOX-1 levels (p < .05) compared to the controls. No significant differences were found between the sLOX-1 level and the degree of coronary atherosclerosis, body mass index, and postmortem interval., Conclusion: sLOX-1 levels in pericardial fluid and urine samples obtained postmortem are useful markers of acute IHD., (Copyright © 2018 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.)

Cholesterol and triglyceride levels have been analyzed in the forensic setting and their values correlated with atherosclerotic lesions found at autopsy and histology. Nevertheless, the results of these investigations have provided diverging information on postmortem molecule stability and postmortem measurement reliability. The aim of this study was to determine triglycerides, total cholesterol, low-density and high-density lipoprotein cholesterol, apolipoprotein B 100 , and apolipoprotein A-I in antemortem and postmortem serum samples in a series of cases (N = 10, including cardiac and noncardiac deaths) that underwent forensic investigations and had both samples available, measure the same molecules in postmortem serum from femoral blood and pericardial and pleural fluids (N = 39, including cardiac and noncardiac deaths), and evaluate whether different levels of these molecules could be observed in cases characterized by different degrees of coronary artery atherosclerosis (N = 39, including cardiac and noncardiac deaths). Preliminary results indicated that total cholesterol and low-density and high-density lipoprotein cholesterol levels in postmortem serum samples tended to be lower than those in antemortem specimens, whereas triglyceride levels in postmortem serum samples tended to be higher than those in antemortem samples. No relationship could be found between postmortem serum and pericardial fluid levels or between postmortem serum and pleural fluid levels of all tested biomarkers. Lastly, cases characterized by severe coronary artery atherosclerosis revealed higher postmortem serum levels of total cholesterol and apolipoprotein B. Globally considered, these data confirm that femoral blood postmortem serum levels of cholesterol and apolipoproteins may be considered suitable to estimate their antemortem values in forensic cases characterized by coronary artery atherosclerosis.

The aim of the present study was to investigate the differential expression of B‑type natriuretic peptide (BNP) between the left and right ventricle (RV) in sudden cardiac death (SCD). A total of 26 forensic autopsy cases of sudden death (survival time <30 min, postmortem interval <48 h or frozen within 6 h following death) in the present institute were examined. The cases consisted of acute ischemic heart disease (AIHD, n=15) with/without apparent myocardial necrosis as a sign of infarction (acute myocardial infarction, n=6; ischemic heart disease, IHD, n=9), and arrhythmogenic right ventricular cardiomyopathy (ARVC/D, n=5), in addition to traffic accidents and high falls without any pre existing heart disease as control (C, total n=6). BNP was investigated in all cases by the colloidal gold method, hematoxylin‑eosin staining, immunohistochemistry (IHC) and the molecular pathological method. The IHC results demonstrated that a positive BNP immunostaining was detected in all groups; however, there was no difference between different causes of death. Pericardial N‑terminal (NT)‑proBNP concentration was significantly increased in deaths resulting from AIHD and ARVC/D compared with control group. The relative quantification of BNP mRNA demonstrated that relative expression levels of BNP mRNA were significantly increased in the left ventricle (LV) in the AIHD group, and in the RV of the ARVC/D group. The relative quantification difference and ratio of BNP mRNA between LV and RV demonstrated a significantly greater value in the AIHD group compared with control group. BNP mRNA in myocardium and NT‑proBNP concentration in pericardial fluid were elevated in SCD patients, and left ventricular dysfunction predominated in AIHD patients, whereas right ventricular dysfunction predominated in ARVC/D patients. The results of the present study suggest the possible use of molecular pathology of BNP for the determination of terminal cardiac function in SCD and analysis of its fatal mechanism in forensic practice.

Background  Neurohumoral and hemodynamic mechanisms have an effect on cardiac activity. C-type natriuretic peptide (CNP) is accessible in the cardiovascular system. The aim of this study was to determine whether CNP concentrations in pericardial fluid and blood are related to cardiac dysfunction in patients undergoing coronary artery bypass graft surgery. Materials and Methods  In this study, 40 patients undergoing coronary artery bypass grafting were enrolled. The patients were separated into two groups according to left ventricular (LV) ejection fraction (EF): group 1 contained 28 patients with normal LV systolic function (LVEF ≥ 50%) and group 2 contained 12 patients with impaired LV systolic function (LVEF < 45%). Plasma and pericardial fluid samples were acquired during surgery to measure CNP levels. Results  In group 1, CNP levels were detected to be 0.46 ± 0.10 ng/mL in plasma and 0.66 ± 0.8 ng/mL in pericardial liquid. In group 2, these levels were 0.51 ± 0.09 and 0.79 ± 0.12 ng/mL, respectively. CNP levels were determined to be significantly higher in patients with low EF compared with those with normal EF in pericardial fluid concentrations ( p  = 0.013). Conclusions  CNP level in pericardial fluid is a more sensitive and proper marker of LV dysfunction than CNP levels in plasma. To the best of our knowledge, this study is the first to examine pericardial fluid CNP levels in patients undergoing coronary artery bypass surgery. It may have a valuable role in organizing cardiac remodeling and hypertrophy., Competing Interests: Funding: The authors declare that there is no conflict of interest. Conflict of Interest This study has not received any grant from any funding agency in the public, commercial, or not-for-profit sectors., (Georg Thieme Verlag KG Stuttgart · New York.)

The pericardial fluid (PF) is contained in the pericardial sac surrounding the heart. MicroRNA (miRNA) exchange via exosomes (endogenous nanoparticles) contributes to cell-to-cell communication. We investigated the hypotheses that the PF is enriched with miRNAs secreted by the heart and that it mediates vascular responses through exosome exchange of miRNAs. The study was developed using leftover material from aortic valve surgery. We found that in comparison with peripheral plasma, the PF contains exosomes enriched with miRNAs co-expressed in patients' myocardium and vasculature. At a functional level, PF exosomes improved survival, proliferation, and networking of cultured endothelial cells (ECs) and restored the angiogenic capacity of ECs depleted (via Dicer silencing) of their endogenous miRNA content. Moreover, PF exosomes improved post-ischemic blood flow recovery and angiogenesis in mice. Mechanistically, (1) let-7b-5p is proangiogenic and inhibits its target gene, TGFBR1, in ECs; (2) PF exosomes transfer a functional let-7b-5p to ECs, thus reducing their TGFBR1 expression; and (3) let-7b-5p depletion in PF exosomes impairs the angiogenic response to these nanoparticles. Collectively, our data support the concept that PF exosomes orchestrate vascular repair via miRNA transfer., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

The aim of this study was to characterize total and specific IgE distribution in postmortem serum as well as pericardial and cerebrospinal fluid samples and evaluate the diagnostic usefulness of total and specific IgE determination in pericardial and cerebrospinal fluids in the forensic setting. Three groups were investigated (non-allergic deaths in non-atopic individuals, fatal allergic anaphylaxis deaths and non-allergic deaths in individuals without medical records). In the first group (non-allergic deaths in non-atopic individuals), total IgE concentrations in postmortem serum from femoral blood, pericardial and cerebrospinal fluids were lower than 40, 32 and 11kU/l, respectively. No specific IgE were identified in any of the sampled fluids. In the second group (fatal allergic anaphylaxis deaths), total IgE concentrations in postmortem serum from femoral blood ranged from 139kU/l to 818kU/l, in pericardial fluid from 89kU/l to 622kU/l and in cerebrospinal fluid from 4kU/l to 11kU/l. A positive Phadiatop ® test and specific IgE antibodies >0.35kU/l were found exclusively in postmortem serum from femoral blood and pericardial fluid. In the third group (non-allergic deaths in individuals without medical records, possibly including atopic individuals), total IgE concentrations ranged from 42kU/l to 516kU/l in postmortem serum from femoral blood, from 34kU/l to 417kU/l in pericardial fluid and from 3kU/l to 12kU/l in cerebrospinal fluid. A positive Phadiatop ® test and specific IgE antibodies >0.35kU/l were found exclusively in postmortem serum from femoral blood and pericardial fluid. These results seem to suggest that total and specific IgE may be measured in postmortem serum from femoral blood and pericardial fluid to estimate total and specific IgE titers at the time of death. Conversely, cerebrospinal fluid total and specific IgE measurement in suspected IgE mediated fatal anaphylaxis cases is of no value for diagnostic purposes., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: The purpose of this study was to examine the relationship between left ventricular (LV) function, cytokine levels and site of myocardial infarction (MI) in patients undergoing coronary artery bypass grafting (CABG)., Methods: Sixty patients undergoing CABG were divided into three groups (n = 20) according to their history of site of myocardial infarction (MI): no previous MI, anterior MI and posterior/inferior MI. In the pre-operative period, detailed analysis of LV function was done by transthoracic echocardiography. The levels of adrenomedullin, interleukin-1-beta, interleukin-6, tumour necrosis factor-alpha (TNF-α) and angiotensin-II in both peripheral blood samples and pericardial fluid were also measured., Results: Echocardiographic analyses showed that the anterior MI group had significantly worse LV function than both the group with no previous MI and the posterior/inferior MI group (p < 0.05 for LV end-systolic diameter, fractional shortening, LV end-systolic volume, LV end-systolic volume index and ejection fraction). In the anterior MI group, both plasma and pericardial fluid levels of adrenomedullin and and pericardial fluid levels of interleukin-6 and interleukin- 1-beta were significantly higher than those in the group with no previous MI (p < 0.05), and pericardial fluid levels of adrenomedullin, interleukin-6 and interleukin-1-beta were significantly higher than those in the posterior/inferior MI group (p < 0.05)., Conclusions: The results of this study indicate that (1) patients with an anterior MI had worse LV function than patients with no previous MI and those with a posterior/inferior MI, and (2) cytokine levels in the plasma and pericardial fluid in patients with anterior MI were increased compared to patients with no previous MI.

Significantly increased blood ketone body levels can be occasionally observed in the forensic setting in situations other than exposure to cold, diabetic or alcoholic ketoacidosis. Though infrequent, these cases do occur and deserve thorough evaluation in order to establish appropriate differential diagnoses and quantify the role that hyperketonemia may play in the death process. Starvation ketoacidosis is a rare cause of metabolic acidosis and is a phenomenon that occurs normally during fasting, as the body switches from carbohydrate to lipid energy sources. The levels of ketonemia in starvation ketoacidosis is usually mild in comparison to those seen in diabetic or alcoholic ketoacidosis. In the clinical setting, several cases of starvation-induced ketoacidosis mainly associated with gastric banding, pregnancy, malnutrition and low-carbohydrate diets have been reported. However, starvation ketosis causing severe metabolic acidosis has been rarely described in the medical literature. In the realm of forensic pathology, starvation-induced hyperketonemia has been rarely described. In this paper we present the postmortem biochemical results observed in situations of suspected starvation-induced hyperketonemia that underwent medico-legal examination. In all these cases, the diagnosis of starvation induced-hyperketonemia and the subsequent ketoacidosis was established per exclusionem based on all postmortem investigation findings. A review of the literature pertaining to the clinical diagnosis of starvation ketoacidosis is also provided., (Copyright © 2016 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.)

The identification and quantitation of the main psychoactive component of Salvia divinorum (salvinorin A) in biological specimens are crucial in forensic and clinical toxicology. Despite all the efforts made, its uncontrolled abuse has increased quickly, exposing its users' health to serious risks both in the short and long term. The use of alternative biological matrices in toxicological analyzes can be advantageous as complementary postmortem samples, or in situations when neither blood nor urine can be collected; they may be useful tools in those determinations, providing important information about prior exposure. The aim of this article is to present a brief summary of legal aspects of Salvia divinorum and salvinorin A, including the methods used for the determination of the latter in biological matrices.

Purpose: The main objective of this study was to test, for the first time, a highly sensitive cardiac troponin T (cTnThs) assay in postmortem serum and pericardial fluid and to evaluate cardiac troponin T (cTnT) levels and their stability after death at different postmortem intervals, in an attempt to determine the viability of the cTnThs assay in the postmortem diagnosis of the cause of death., Methods: cTnT levels were determined in serum and pericardial fluid samples taken from 58 cadavers at known postmortem intervals, whose causes of death were categorized into the following groups: (1) sudden cardiac deaths, (2) multiple trauma, (3) mechanical asphyxia, and (4) other natural deaths. cTnT was determined by inmunoassay, using the Troponin T highly sensitive STAT assay (Roche(®))., Results: Average cTnT levels measured by a highly sensitive assay in postmortem serum were markedly higher than clinical serum levels. Moreover, similar results, higher cTnT levels in postmortem pericardial fluid, were obtained when compared to levels found in pericardial fluid taken from two living patients during coronary artery bypass surgery. cTnT levels in both postmortem fluids remained stable for up to 34 h after death. No differences in cTnT levels in either postmortem fluid by sex and age were detected. Levels of cTnT found in pericardial fluid in the other natural deaths group were significantly lower than the cTnT levels found in that postmortem fluid from any of the other causes of death groups., Conclusion: It is therefore reasonable to conclude that determination of cTnT by a highly sensitive assay in pericardial fluid can provide forensic pathologists with a complementary test to the diagnosis of cause of death.

Aim: Obesity and especially hypertrophy of epicardial adipose tissue accelerate coronary atherogenesis. We aimed at comparing levels of inflammatory and atherogenic hormones from adipose tissue in the pericardial fluid and circulation of cardiovascular disease patients., Methods and Results: Venous plasma (P) and pericardial fluid (PF) were obtained from elective cardiothoracic surgery patients (n = 37). Concentrations of leptin, adipocyte fatty acid-binding protein (A-FABP) and adiponectin (APN) were determined by enzyme-linked immunosorbent assays (ELISA). The median concentration of leptin in PF (4.3 (interquartile range: 2.8-9.1) μg/L) was comparable to that in P (5.9 (2.2-11) μg/L) and these were significantly correlated to most of the same patient characteristics. The concentration of A-FABP was markedly higher (73 (28-124) versus 8.4 (5.2-14) μg/L) and that of APN was markedly lower (2.8 (1.7-4.2) versus 13 (7.2-19) mg/L) in PF compared to P. APN in PF was unlike in P not significantly related to age, body mass index, plasma triglycerides or coronary artery disease. PF levels of APN, but not A-FABP, were related to the size of paracardial adipocytes. PF levels of APN and A-FABP were not related to the immunoreactivity of paracardial adipocytes for these proteins., Conclusion: In cardiac and vascular disease patients, PF is enriched in A-FABP and poor in APN. This adipokine microenvironment is more likely determined by the heart than by the circulation or paracardial adipose tissue.

Background: Recently, it has been reported that specific microRNA (miRNA) levels are elevated in serum and can be used as biomarkers in patients with cardiovascular diseases. However, miRNAs expression profiles and their sources in pericardial fluid (PF) are unclear., Methods and Results: The purpose of this study was to identify the levels of miRNAs in PF in relation to those in the serum in patients undergoing cardiac surgery. Serum (S) and PF from patients undergoing coronary artery bypass graft (CABG) due to stable angina pectoris (sAP) and unstable AP (uAP) and aortic valve replacement due to aortic stenosis (AS) were analyzed for the detection of miRNAs. We named these samples S-sAP, S-uAP, S-AS, PF-sAP, PF-uAP, and PF-AS, respectively. We first measured the levels of miR-423-5p, which was recognized previously as a biomarker for heart failure. miR-423-5p levels were significantly higher in PF than serum. Although there was no difference in miR-423-5p levels among the PF-AS, PF-sAP, and PF-uAP, its levels were significantly elevated in S-uAP compared with those in S-AS and S-sAP. In order to clarify the source of miR-423-5p in PF, we measured the levels of muscle-enriched miR-133a and vascular-enriched miR-126 and miR-92a in the same samples. miR-133a levels were significantly higher in serum than in PF, and it was elevated in S-uAP compared with S-AS. miR-126 level was significantly increased in serum compared with PF, and the level of miR-92a the similar tendency. miR-423-5p is located in the first intron of NSRP1. There is another miRNA, miR-3184, encoded in the opposite direction in the same region. In vitro experiments indicated that the duplex of miR-423-5p and miR-3184-3p was more resistant to RNase than the duplex of miR-423-5p and miR-133-3p, which may help to stabilize miR-423-5p in the PF., Conclusions: Our results suggested that miR-423-5p is enriched in PF, and serum miR-423-5p may be associate with uAP. Its expression pattern was different to that of muscle- and vascular-enriched miRNAs, miR-133a, miR-126, and miR-92a.

Background: Pericardial effusions and acute renal failure are common findings in clinical practice. However, acute renal failure resulting from pericardial effusions (without tamponade) is a rare finding. We report the first such case to occur in a transplanted kidney., Case Presentation: A 20-year-old Sri Lankan male presented with hypertensive crisis in the background of end stage renal failure. He was thoroughly investigated for secondary causes of hypertension to no avail. He was hemodialysed adequately for 6 months, while being worked up for transplantation. He received an ABO matched, living donor transplant. Immediate post-operative period his urine outputs were poor, soon to became anuric by 6 h post-transplant. Elevated liver enzymes and non-specific increase of resistivity indexes (0.84-0.88) at the Doppler scan raised the possibility of venous hypertension. An echocardiogram showed a moderately large pericardial effusion which was tapped, and found to be a transudate. He started producing urine within 6 h, entered polyuric phase by day 3, and by day 7 his creatinine dropped to reference levels. Vasculitis screen, anti nuclear factor, viral screen, and rickettsia serology were negative. Albumin levels on day 2 were 27 g/l and were replaced using human albumin. The exact cause of pericardial effusion is unclear but hypoalbuminemia, drug-induced and idiopathic are possible causes. He has excellent graft function, no recurrences or constrictive pericarditis after 2 years follow., Conclusion: We recommend any patient who has delayed graft function and raised central venous pressures to have an echocardiogram to exclude pericardial effusions. The response to pericardiocentesis had been universally good in reported cases.

Background: We recently demonstrated that epicardial progenitor cells participate in the regenerative response to myocardial infarction (MI) and factors released in the pericardial fluid (PF) may play a key role in this process. Exosomes are secreted nanovesicles of endocytic origin, identified in most body fluids, which may contain molecules able to modulate a variety of cell functions. Here, we investigated whether exosomes are present in the PF and their potential role in cardiac repair., Methods and Results: Early gene expression studies in 3day-infarcted mouse hearts showed that PF induces epithelial-to-mesenchymal transition (EMT) in epicardial cells. Exosomes were identified in PFs from non-infarcted patients (PFC) and patients with acute MI (PFMI). A shotgun proteomics analysis identified clusterin in exosomes isolated from PFMI but not from PFC. Notably, clusterin has a protective effect on cardiomyocytes after acute MI in vivo and is an important mediator of TGFβ-induced. Clusterin addition to the pericardial sac determined an increase in epicardial cells expressing the EMT marker α-SMA and, interestingly, an increase in the number of epicardial cells ckit(+)/α-SMA(+), 7days following MI. Importantly, clusterin treatment enhanced arteriolar length density and lowered apoptotic rates in the peri-infarct area. Hemodynamic studies demonstrated an improvement in cardiac function in clusterin-treated compared to untreated infarcted hearts., Conclusions: Exosomes are present and detectable in the PFs. Clusterin was identified in PFMI-exosomes and might account for an improvement in myocardial performance following MI through a framework including EMT-mediated epicardial activation, arteriogenesis and reduced cardiomyocyte apoptosis., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background: Pericardial fluid (PF) contains several biologically active substances, which may provide information regarding the cardiac conditions. Nitric oxide (NO) has been implicated in cardiac remodeling. We hypothesized that L-arginine (L-Arg) precursor of NO-synthase (NOS) and asymmetric dimethylarginine (ADMA), an inhibitor of NOS, are present in PF of cardiac patients and their altered levels may contribute to altered cardiac morphology., Methods: L-Arg and ADMA concentrations in plasma and PF, and echocardiographic parameters of patients undergoing coronary artery bypass graft (CABG, n = 28) or valve replacement (VR, n = 25) were determined., Results: We have found LV hypertrophy in 35.7% of CABG, and 80% of VR patients. In all groups, plasma and PF L-Arg levels were higher than that of ADMA. Plasma L-Arg level was higher in CABG than VR (75.7 ± 4.6 μmol/L vs. 58.1 ± 4.9 μmol/L, p = 0.011), whereas PF ADMA level was higher in VR than CABG (0.9 ± 0.0 μmol/L vs. 0.7 ± 0.0 μmol/L, p = 0.009). L-Arg/ADMA ratio was lower in the VR than CABG (VRplasma: 76.1 ± 6.6 vs. CABGplasma: 125.4 ± 10.7, p = 0.004; VRPF: 81.7 ± 4.8 vs. CABGPF: 110.4 ± 7.2, p = 0.009). There was a positive correlation between plasma L-Arg and ADMA in CABG (r = 0.539, p = 0.015); and plasma and PF L-Arg in CABG (r = 0.357, p = 0.031); and plasma and PF ADMA in VR (r = 0.529, p = 0.003); and PF L-Arg and ADMA in both CABG and VR (CABG: r = 0.468, p = 0.006; VR: r = 0.371, p = 0.034). The following echocardiographic parameters were higher in VR compared to CABG: interventricular septum (14.7 ± 0.5 mm vs. 11.9 ± 0.4 mm, p = 0.000); posterior wall thickness (12.6 ± 0.3 mm vs. 11.5 ± 0.2 mm, p = 0.000); left ventricular (LV) mass (318.6 ± 23.5 g vs. 234.6 ± 12.3 g, p = 0.007); right ventricular (RV) (33.9 ± 0.9 cm2 vs. 29.7 ± 0.7 cm2, p = 0.004); right atrial (18.6 ± 1.0 cm2 vs. 15.4 ± 0.6 cm2, p = 0.020); left atrial (19.8 ± 1.0 cm2 vs. 16.9 ± 0.6 cm2, p = 0.033) areas. There was a positive correlation between plasma ADMA and RV area (r = 0.453, p = 0.011); PF ADMA and end-diastolic (r = 0.434, p = 0.015) and systolic diameter of LV (r = 0.487, p = 0.007); and negative correlation between PF ADMA and LV ejection fraction (r = -0.445, p = 0.013) in VR., Conclusion: We suggest that elevated levels of ADMA in the PF of patients indicate upregulated RAS and reduced bioavailability of NO, which can contribute to the development of cardiac hypertrophy and remodeling.

Monitoring the bioenergetics of leucocytes is now emerging as an important approach in translational research to detect mitochondrial dysfunction in blood or other patient samples. Using the mitochondrial stress test, which involves the sequential addition of mitochondrial inhibitors to adherent leucocytes, we have calculated a single value, the Bioenergetic Health Index (BHI), which represents the mitochondrial function in cells isolated from patients. In the present report, we assess the BHI of monocytes isolated from the post-operative blood and post-operative pericardial fluid (PO-PCF) from patients undergoing cardiac surgery. Analysis of the bioenergetics of monocytes isolated from patients' PO-PCF revealed a profound decrease in mitochondrial function compared with monocytes isolated from their blood or from healthy controls. Further, patient blood monocytes showed no significant difference in the individual energetic parameters from the mitochondrial stress test but, when integrated into the BHI evaluation, there was a significant decrease in BHI compared with healthy control monocytes. These data support the utility of BHI measurements in integrating the individual parameters from the mitochondrial stress test into a single value. Supporting our previous finding that the PO-PCF is pro-oxidant, we found that exposure of rat cardiomyocytes to PO-PCF caused a significant loss of mitochondrial membrane potential and increased reactive oxygen species (ROS). These findings support the hypothesis that integrated measures of bioenergetic health could have prognostic and diagnostic value in translational bioenergetics., (© 2015 Authors.)

Aims: Multicellular organisms maintain vital functions through intercellular communication. Release of extracellular vesicles that carry signals to even distant target organs is one way of accomplishing this communication. MicroRNAs can also be secreted from the cells in exosomes and act as paracrine signalling molecules. In addition, microRNAs have been implicated in the pathogenesis of a large number of diseases, including cardiovascular diseases, and are considered as promising candidate biomarkers due to their relative stability and easy quantification from clinical samples. Pericardial fluid contains hormones secreted by the heart and is known to reflect the cardiac function. In this study, we sought to investigate whether pericardial fluid contains microRNAs and if so, whether they could be used to distinguish between different cardiovascular pathologies and disease stages., Methods and Results: Pericardial fluid was collected from heart failure patients during open-heart surgery. MicroRNA profiles of altogether 51 patients were measured by quantitative real-time PCR (qPCR) using Exiqon human panels I and II. On the average, 256 microRNAs were detected per sample, and 70 microRNAs out of 742 profiled microRNAs were detected in every sample. The five most abundant microRNAs in pericardial fluid were miR-21-5p, miR-451a, miR-125b-5p, let-7b-5p and miR-16-5p. No specific signatures for cardiovascular pathologies or clinically assessed heart failure stages could be detected from the profiles and, overall, microRNA profiles of the samples were found to be very similar despite the heterogeneity in the study population., Conclusion: Measured microRNA profiles did not separate the samples according to the clinical features of the patients. However, several previously identified heart failure marker microRNAs were detected. The pericardial fluid microRNA profile appeared to be a result of an active and selective secretory process indicating that microRNAs may act as paracrine signalling factors by mediating the local crosstalk between cardiac cells.

Objective: Cytospin preparations and immunocytochemistry are common methods in hospitals to evaluate malignancies in body fluids. Characteristics of malignant cells in pediatric body fluids have not been adequately evaluated., Study Design: 183 pleural, peritoneal and pericardial pediatric fluid specimens were examined by cytospin preparations and immunocytochemistry from two hospitals using similar procedural techniques. Cytologic diagnoses were correlated with the results of clinical history, histology and ancillary studies., Results: Forty cases with malignancy were identified (21.9%); the most common diagnoses were rhabdomyosarcoma and acute lymphoblastic lymphoma (9 and 8 cases, respectively). Small round cell tumors revealed similar morphology as clusters of small round cells with central nuclei and scant cytoplasm with frequent small vacuoles. Twenty-one cases were evaluated by immunocytochemistry, 12 by flow cytometry and 5 by cytogenetic analysis. CD3, CD20, TdT, CD10, desmin and myogenin were the most common markers. Staining artifacts causing interpretation difficulties were noted in 5 cases that were resolved by molecular studies and deferral for surgical specimens., Conclusions: Small round cell tumors are the most common malignancies encountered in pediatric body fluids and share a nonspecific morphology. Although immunocytochemistry is helpful to arrive at the correct diagnosis, other ancillary studies may be necessary, particularly in hematologic malignancies and other difficult cases., (© 2015 S. Karger AG, Basel.)