Second opinion system for sudden cardiac death cases in forensic practice.

Sudden cardiac death (SCD) represents a considerable percentage of cardiovascular deaths worldwide. The most frequent pathological substrate of SCD is atherosclerotic coronary artery disease (CAD). The other, less common, pathologies which can cause SCD include cardiomyopathies, congenital diseases (including abnormal anatomy), and arrhythmias such as channelopathies, many of which are genetically determined. Autopsies of SCD victims are generally performed by forensic pathologists. In some cases, a third person responsibility could be invoked. While CAD diagnosis at post-mortem examination is not a major challenge for the forensic pathologist, the other rarer diseases may be. In such instances, referral of the hearts to specialized centers with recognized expertise is recommended, and this is particularly important in cases of SCDs of young people. Moreover, in order to avoid the frequent overdiagnosis of a pathological heart, an expert opinion should be sought for even in the presence of a morphologically normal heart. In cases where retention of the heart is not feasible, it is essential to provide an extensive photographic documentation, with the indication of the sampling sites for histological examination. However, some practical aspects, as the criteria for case selection in routine forensic practice are missing. In this paper, we present the recommendations for heart retention for a second expert opinion and the alternative of documentation and sampling for cases where retention is not possible.

Death by anaphylactic shock in an institution: an accident or negligence?

This is a description of a man, institutionalised for learning difficulties, known to have an allergy to seafood. After eating a pie, the patient quickly developed dyspnoea and vomiting. The staff at the institution administered epinephrine and called the emergency services. Despite cardiopulmonary resuscitation, the patient died shortly after being admitted to the emergency department of the University Hospitals of Geneva. In the light of the circumstances of the death and of a discrepancy between the information given to the police by the staff at the institution looking after the patient on the one hand, and the preliminary elements of the investigation on the other hand, it was suspected that there was failure in care of the patient and our institute was asked to carry out an autopsy. Basing on all the investigations carried out, the cause of death was anaphylactic reaction following the ingestion of seafood, contrary to what had been alleged by the staff at the home.

Molecular tissue changes in early myocardial ischemia: from pathophysiology to the identification of new diagnostic markers.

Diagnosing early myocardial ischemia (the initial 4 to 6 h after interruption of blood flow to part of the myocardium) remains a challenge for clinical and forensic pathologists. Several immunohistochemical markers have been proposed for improving postmortem detection of early myocardial ischemia; however, no single marker appears to be both sufficiently specific as well as sensitive. This review summarizes the diverse categories of molecular tissue markers that have been investigated in human autopsy samples with acute myocardial infarction as well as in the well-established and widely used in vivo animal model of early myocardial ischemia (permanent ligation of the coronary artery). Recently identified markers appearing during the initial 2 h of myocardial ischemia are highlighted. Among them, only six were tested for specificity (C5b-9, hypoxia-inducible factor 1-alpha, vascular endothelial growth factor, heart fatty acid binding protein, connexin 43, and JunB). Despite the discovery of several potentially promising markers (in terms of early expression and specificity), many of them remain to be tested and validated for application in routine diagnostics in clinical and forensic pathology. In particular, research investigating the postmortem stability of these markers is required before any might be implemented into routine diagnostics. Establishing a standardized panel of immunohistochemical markers may be more useful for improving sensitivity and specificity than searching for a single marker.

Early markers of myocardial ischemia: from the experimental model to forensic pathology cases of sudden cardiac death.

The goal of this study was to assess whether early markers of myocardial ischemia, identified in a previous experimental work, can be applied in forensic pathology cases of sudden, ischemic cardiac death. These markers include desphosphorylated connexin 43 (Cx43), JunB, TUNEL assay, myoglobin, and troponin T. Fourteen cases of sudden cardiac death with gross and/or histological signs of myocardial infarction and 14 cases of sudden cardiac death with signs of early ischemia at histology and positive immunoreactions for fibronectin and C5b-9 were investigated. The control group was represented by 15 hanging (global hypoxia) cases. Immunohistochemical reactions were classified into four degrees and compared among groups. Cx43 and JunB were significantly more expressed in hanging than in ischemia/infarction, but they showed a different distribution in the tissue (sub-endocardial in ischemia/infarction, diffuse in hanging) and a different intensity of the signal. TUNEL assay was significantly more expressed in the group of early ischemia than in myocardial infarction. Myoglobin and troponin T did not show any significantly different expression among the three groups. Depletion markers have a limited application in forensic cases, and this is mostly because positive (depleted) areas are difficult to distinguish from artifactually paler areas. Nuclear markers (JunB and TUNEL), on the other hand, require a well-trained eye and a high magnification in order to be distinguished. Cx43, JunB, and TUNEL assays were confirmed to be early, sensitive markers for myocardial ischemia. Nonetheless, they are not specific, as they are expressed in global hypoxia as well, but with a different tissular distribution.

Multiplex quantitative imaging of human myocardial infarction by mass spectrometry-immunohistochemistry.

Simultaneous assessment of a panel of protein markers is becoming essential in order to enhance biomarker research and improve diagnostics. Specifically, postmortem diagnostics of early myocardial ischemia in sudden cardiac death cases could benefit from a multiplex marker assessment in the same tissue section. Current analytical antibody-based techniques (immunohistochemistry and immunofluorescence) limit multiplex analysis usually to not more than three antibodies. In this study, mass spectrometry-immunohistochemistry (MS-IHC) was performed by combining laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) with rare-metal-isotope-tagged antibodies as a technique for multiplex analysis of human postmortem myocardial tissue samples. Tissue sections with myocardial infarction were simultaneously analyzed for seven primary, rare-metal-isotope-tagged antibodies (troponin T, myoglobin, fibronectin, C5b-9, unphosphorylated connexin 43, VEGF-B, and JunB). Comparison between the MS-IHC approach and chromogenic IHC showed similar patterns in ionic and optical images. In addition, absolute quantification was performed by MS-IHC, providing a proportional relationship between the signal intensity and the local marker concentration in tissue sections. These data demonstrated that LA-ICP-MS combined with rare-metal-isotope-tagged antibodies is an efficient strategy for simultaneous testing of multiple markers and allows not only visualization of molecules within the tissue but also quantification of the signal. Such imaging approach has a great potential in both diagnostics and pathology-related research.

Visualization of Myocardial Infarction in Postmortem Multiphase Computed Tomography Angiography: A Feasibility Study.

Recent studies have indicated that multiphase postmortem computed tomography angiography (MPMCTA) allows detection of a pathological enhancement of the myocardium in regions that correlate with the localization of the infarction at histology. The aim of this study was to verify this hypothesis by examining MPMCTA images in cases of myocardial infarction. Therefore, we investigated 10 autopsy cases where death was attributed to myocardial infarction or which showed cardiovascular pathology. As a control group, we selected 10 cases of non-natural (namely, not cardiac) death. The MPMCTA was performed in both groups to ascertain whether a pathological enhancement could be observed. We detected a myocardial enhancement in all cardiac death cases, in the same region that showed infarction at histology. No enhancement was observed in control cases. These results have important implications in the routine management of sudden cardiac death cases. In fact, MPMCTA can not only orient about the cause of death before autopsy, but can especially help to identify affected regions for guiding and improving the sampling for microscopic examination.

Technical note: EnVision™ FLEX improves the detectability of depletions of myoglobin and troponin T in forensic cases of myocardial ischemia/infarction.

Immunohistochemistry is a well-established technique used in many research laboratories as well as in clinical diagnostics. The method allows to visualize the expression of proteins in biological tissues, as well as to evaluate this expression semi-quantitatively. For diagnosis, an optimal staining, based on a straightforward protocol, is crucial. In many sudden cardiac death cases, immunohistochemistry is the only tool enabling the diagnosis of myocardial ischemia/infarction, thus the diagnosis of the cause of death. Improvements in immunoreactions are actually possible thanks to optimized detection systems. The recently introduced detection system EnVision Flex™ by Dako allows to dramatically improve (in terms of intensity of the signal and practically absence of background) the visualization of antigens in formalin-fixed paraffin-embedded (FFPE) tissue sections. We tested this method for the detection of myoglobin and troponin T in human postmortem cases of myocardial infarction, as the results obtained by using the « classical ¼ ABC (avidin-biotin complex) method have proven to be sub-optimal, thus rendering any interpretation very difficult, if not impossible.

Early markers for myocardial ischemia and sudden cardiac death.

The post-mortem diagnosis of acute myocardial ischemia remains a challenge for both clinical and forensic pathologists. We performed an experimental study (ligation of left anterior descending coronary artery in rats) in order to identify early markers of myocardial ischemia, to further apply to forensic and clinical pathology in cases of sudden cardiac death. Using immunohistochemistry, Western blots, and gene expression analyses, we investigated a number of markers, selected among those which are currently used in emergency departments to diagnose myocardial infarction and those which are under investigation in basic research and autopsy pathology studies on cardiovascular diseases. The study was performed on 44 adult male Lewis rats, assigned to three experimental groups: control, sham-operated, and operated. The durations of ischemia ranged between 5 min and 24 h. The investigated markers were troponins I and T, myoglobin, fibronectin, C5b-9, connexin 43 (dephosphorylated), JunB, cytochrome c, and TUNEL staining. The earliest expressions (≤30 min) were observed for connexin 43, JunB, and cytochrome c, followed by fibronectin (≤1 h), myoglobin (≤1 h), troponins I and T (≤1 h), TUNEL (≤1 h), and C5b-9 (≤2 h). By this investigation, we identified a panel of true early markers of myocardial ischemia and delineated their temporal evolution in expression by employing new technologies for gene expression analysis, in addition to traditional and routine methods (such as histology and immunohistochemistry). Moreover, for the first time in the autopsy pathology field, we identified, by immunohistochemistry, two very early markers of myocardial ischemia: dephosphorylated connexin 43 and JunB.

Anterior Petrosectomy With Intertentorial Approach.

BACKGROUND AND OBJECTIVES: The extradural anterior petrosal approach (EAPA) can present a challenge because it deals with critical structures in a narrow, confined corridor. It is associated with several potential approach-related risks including temporal lobe and venous injuries. Tentorial peeling has the potential to largely eliminate these risks during the approach and may offer more options for tailoring the dural opening to the anatomic region that one wants to expose. METHODS: Anatomic dissections of five adult injected non-formalin-fixed cadaveric heads were performed. Anterior petrosectomy with intertentorial approach (APIA) through a tentorial peeling was completed. Step-by-step documentation of the cadaveric dissections and diagrammatic representations are presented along with an illustrative case. RESULTS: Tentorial peeling separates the tentorium into a temporal tentorial leaf and posterior fossa tentorial leaf, adding a fourth dural layer to the three classic ones described during a standard EAPA. This opens out the intertentorial space and offers more options for tailoring the dural incisions specific to the pathology being treated. This represents a unique possibility to address brainstem or skull base pathology along the mid- and upper clivus with the ability to keep the entire temporal lobe and basal temporal veins covered by the temporal tentorial leaf. The APIA was successfully used for the resection of a large clival chordoma in the illustrative case. CONCLUSION: APIA is an interesting modification to the classic EAPA to reduce the approach-related morbidity. The risk reduction achieved is by eliminating the exposure of the temporal lobe while maintaining the excellent access to the petroclival region. It also provides several options to tailor the durotomies based on the localization of the lesion.

Combined Petrosal Intertentorial Approach: A Cadaveric Study of Comparison With the Standard Combined Petrosectomy.

BACKGROUND AND OBJECTIVES: The combined petrosal intertentorial approach (CPIA) has been proposed as an alternative to standard combined petrosal approach (SCPA). CPIA has been designed to maintain integrity of the temporal dura with a view to reduce temporal lobe morbidity and venous complications. This study has been designed to perform a quantitative comparison between these approaches. METHODS: Five human specimens were used for this study. CPIA was performed on one side and SCPA on the opposite side. The area of exposure (petroclival and brainstem), surgical freedom, and angles of attack to a predefined target were measured and compared. RESULTS: SCPA provided a significantly larger petroclival area of exposure (6.81 ± 0.60 cm2) over the CPIA (5.59 ± 0.59 cm2), P = .012. The area of brainstem exposed with SCPA was greater than with CPIA (7.17 ± 0.84 vs 5.63 ± 0.72, P = .014). The area of surgical freedom was greater in SCPA rather than in CPIA (8.59 ± 0.55 and 7.13 ± 0.96 cm2, respectively, P = .019). There was no significative difference between CPIA and SCPA in the vertical angles of attack for the Meckel cave, Dorello canal, and root entry zone of cranial nerve VII. Conversely, the horizontal angles of attack permitted by the CPIA were significantly smaller for the Meckel cave (52.36° ± 5.01° vs 64.4° ± 5.3°, P = .006) and root entry zone of cranial nerve VII (30.7° ± 4.4° vs 40.1° ± 6.2°, P = .025). CONCLUSION: CPIA is associated with a reduction in terms of the area of surgical freedom (22%), skull base (18%), brainstem exposure (17%), and horizontal angles of attack (18%-23%) when compared with SCPA. This loss in terms of exposure is counterbalanced by the advantage of keeping the temporal lobe covered by an extra layer of meningeal tissue, thus possibly reducing the risk of temporal lobe injury and venous infarction. These results need to be validated with adequate clinical experience.

Sensitivity and specificity of NT-proBNP to detect heart failure at post mortem examination.

NT-proBNP, a marker of cardiac failure, has been shown to be stable in post mortem samples. The aim of this study was to assess the accuracy of NT-proBNP to detect heart failure in the forensic setting. One hundred sixty-eight consecutive autopsies were included in the study. NT-proBNP blood concentrations were measured using a chemiluminescent immunoassay kit. Cardiac failure was assessed by three independent forensic experts using macro- and microscopic findings complemented by information about the circumstances of body discovery and the known medical story. Area under the receiving operator curve was of 65.4% (CI 95%, from 57.1 to 73.7). Using a standard cut-off value of >220 pg/mL for NT-proBNP blood concentration, heart failure was detected with a sensitivity of 50.7% and a specificity of 72.6%. NT-proBNP vitreous humor values were well correlated to the ones measured in blood (r (2) = 0.658). Our results showed that NT-proBNP can corroborate the pathological findings in cases of natural death related to heart failure, thus, keeping its diagnostic properties passing from the ante mortem to the post mortem setting. Therefore, biologically inactive polypeptides like NT-proBNP seem to be stable enough to be used in forensic medicine as markers of cardiac failure, taking into account the sensitivity and specificity of the test.

Transient and Efficient Vascular Permeability Window for Adjuvant Drug Delivery Triggered by Microbeam Radiation.

BACKGROUND: Microbeam Radiation Therapy (MRT) induces a transient vascular permeability window, which offers a novel drug-delivery system for the preferential accumulation of therapeutic compounds in tumors. MRT is a preclinical cancer treatment modality that spatially fractionates synchrotron X-rays into micrometer-wide planar microbeams which can induce transient vascular permeability, especially in the immature tumor vessels, without compromising vascular perfusion. Here, we characterized this phenomenon using Chicken Chorioallantoic Membrane (CAM) and demonstrated its therapeutic potential in human glioblastoma xenografts in mice. METHODS: the developing CAM was exposed to planar-microbeams of 75 Gy peak dose with Synchrotron X-rays. Similarly, mice harboring human glioblastoma xenografts were exposed to peak microbeam doses of 150 Gy, followed by treatment with Cisplatin. Tumor progression was documented by Magnetic Resonance Imaging (MRI) and caliper measurements. RESULTS: CAM exposed to MRT exhibited vascular permeability, beginning 15 min post-irradiation, reaching its peak from 45 min to 2 h, and ending by 4 h. We have deemed this period the "permeability window". Morphological analysis showed partially fragmented endothelial walls as the cause of the increased transport of FITC-Dextran into the surrounding tissue and the extravasation of 100 nm microspheres (representing the upper range of nanoparticles). In the human glioblastoma xenografts, MRI measurements showed that the combined treatment dramatically reduced the tumor size by 2.75-fold and 5.25-fold, respectively, compared to MRT or Cisplatin alone. CONCLUSIONS: MRT provides a novel mechanism for drug delivery by increasing vascular transpermeability while preserving vessel integrity. This permeability window increases the therapeutic index of currently available chemotherapeutics and could be combined with other therapeutic agents such as Nanoparticles/Antibodies/etc.

Detecting early myocardial ischemia in rat heart by MALDI imaging mass spectrometry.

Diagnostics of myocardial infarction in human post-mortem hearts can be achieved only if ischemia persisted for at least 6-12 h when certain morphological changes appear in myocardium. The initial 4 h of ischemia is difficult to diagnose due to lack of a standardized method. Developing a panel of molecular tissue markers is a promising approach and can be accelerated by characterization of molecular changes. This study is the first untargeted metabolomic profiling of ischemic myocardium during the initial 4 h directly from tissue section. Ischemic hearts from an ex-vivo Langendorff model were analysed using matrix assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) at 15 min, 30 min, 1 h, 2 h, and 4 h. Region-specific molecular changes were identified even in absence of evident histological lesions and were segregated by unsupervised cluster analysis. Significantly differentially expressed features were detected by multivariate analysis starting at 15 min while their number increased with prolonged ischemia. The biggest significant increase at 15 min was observed for m/z 682.1294 (likely corresponding to S-NADHX-a damage product of nicotinamide adenine dinucleotide (NADH)). Based on the previously reported role of NAD+/NADH ratio in regulating localization of the sodium channel (Nav1.5) at the plasma membrane, Nav1.5 was evaluated by immunofluorescence. As expected, a fainter signal was observed at the plasma membrane in the predicted ischemic region starting 30 min of ischemia and the change became the most pronounced by 4 h. Metabolomic changes occur early during ischemia, can assist in identifying markers for post-mortem diagnostics and improve understanding of molecular mechanisms.

Diagnosis of myocardial infarction at autopsy: AECVP reappraisal in the light of the current clinical classification.

Ischemic heart disease is one of the leading causes of morbidity and death worldwide. Consequently, myocardial infarctions are often encountered in clinical and forensic autopsies, and diagnosis can be challenging, especially in the absence of an acute coronary occlusion. Precise histopathological identification and timing of myocardial infarction in humans often remains uncertain while it can be of crucial importance, especially in a forensic setting when third person involvement or medical responsibilities are in question. A proper post-mortem diagnosis requires not only up-to-date knowledge of the ischemic coronary and myocardial pathology, but also a correct interpretation of such findings in relation to the clinical scenario of the deceased. For these reasons, it is important for pathologists to be familiar with the different clinically defined types of myocardial infarction and to discriminate myocardial infarction from other forms of myocardial injury. This article reviews present knowledge and post-mortem diagnostic methods, including post-mortem imaging, to reveal the different types of myocardial injury and the clinical-pathological correlations with currently defined types of myocardial infarction.

Postmortem imaging as a complementary tool for the investigation of cardiac death.

In the past 2 decades, modern radiological methods, such as multiple detector computed tomography (MDCT), MDCT-angiography, and cardiac magnetic resonance imaging (MRI) were introduced into postmortem practice for investigation of sudden death (SD), including cases of sudden cardiac death (SCD). In forensic cases, the underlying cause of SD is most frequently cardiovascular with coronary atherosclerotic disease as the leading cause. There are many controversies about the role of postmortem imaging in establishing the cause of death and especially the value of minimally invasive autopsy techniques. This paper discusses the state of the art for postmortem radiological evaluation of the heart compared to classical postmortem examination, especially in cases of SCD. In SCD cases, postmortem CT is helpful to estimate the heart size and to visualize haemopericardium and calcified plaques and valves, as well as to identify and locate cardiovascular devices. Angiographic methods are useful to provide a detailed view of the coronary arteries and to analyse them, especially regarding the extent and location of stenosis and obstruction. In postsurgical cases, it allows verification and documentation of the patency of stents and bypass grafts before opening the body. Postmortem MRI is used to investigate soft tissues such as the myocardium, but images are susceptible to postmortem changes and further work is necessary to increase the understanding of these radiological aspects, especially of the ischemic myocardium. In postsurgery cases, the value of postmortem imaging of the heart is reportedly for the diagnostic and documentation purposes. The implementation of new imaging methods into routine postmortem practice is challenging, as it requires not only an investment in equipment but, more importantly, investment in the expertise of interpreting the images. Once those requirements are implemented, however, they bring great advantages in investigating cases of SCD, as they allow documentation of the body, orientation of sampling for further analyses and gathering of other information that cannot be obtained by conventional autopsy such as a complete visualization of the vascular system using postmortem angiography.Key pointsThere are no established guidelines for the interpretation of postmortem imaging examination of the heartAt present, postmortem imaging methods are considered as less accurate than the autopsy for cardiac deathsPostmortem imaging is useful as a complementary tool for cardiac deathsThere is still a need to validate postmortem imaging in cardiac deaths by comparing with autopsy findings.

[Genetic identification of dead persons: which reference sample should be used?]. / Identification génétique de personnes défuntes: quel echantillon de référence choisir?

Identification of a deceased person consists in connecting this person with reference data. Within this context, DNA analyses allow to use samples coming from the deceased himself (personal reference) or from persons closely related to the deceased (familial reference). The analysis of 132 genetic identifications performed between 2003 and 2007 in Switzerland illustrates that familial references are predominantly used. Recommendations are presented to optimize the genetic identification process. In particular, personal references collected on the deceased when alive should be preferred. When this is not possible, several reference samples should be analysed in order to minimize the probability of a fortuitous connection.

Recommendations for genetic testing and counselling after sudden cardiac death: practical aspects for Swiss practice.

There is a need to standardise, within a coordinated Swiss framework, the practical aspects of genetic testing and genetic counselling on possibly inherited cardiovascular disorders in relatives of a sudden cardiac death (SCD) victim. Because of the major advances in genetic investigation techniques and recent publication of international guidelines in the field of cardiology, genetics and pathology, we consider it important to summarise the current evidence and propose an optimal approach to post-mortem genetic investigation for SCD victims and their families in Switzerland. In this article, we discuss important technical, financial and medico-ethical aspects, and provide updated information on specific situations in which forensic pathologists, general practitioners and cardiologists should suspect a genetic origin of the SCD. At present, the principles of benefit, the duty to warn and the impact of genetic information for family members at risk are considered as strong justifications for post-mortem disclosure and prevail over the arguments of respect for a deceased person's privacy and confidentiality. This paper underlines also the need to update and improve the general knowledge concerning the genetic risk of cardiovascular pathologies, the importance to perform an autopsy and post-mortem genetic testing in SCD victims, and to develop standardized post-mortem disclosure policy at national and international levels for SCD cases and relatives.

Postmortem biochemistry in suspected starvation-induced ketoacidosis.

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.

Post-mortem determination of insulin using chemiluminescence enzyme immunoassay: preliminary results.

Insulin determination in blood sampled during post-mortem investigation has been repeatedly asserted as being of little diagnostic value due to the rapid occurrence of decompositional changes and blood haemolysis. In this study, we assessed the feasibility of insulin determination in post-mortem serum, vitreous humour, bile, and cerebrospinal and pericardial fluids in one case of fatal insulin self-administration and a series of 40 control cases (diabetics and non-diabetics) using a chemiluminescence enzyme immunoassay. In the case of suicide by insulin self-administration, insulin concentrations in pericardial fluid and bile were higher than blood clinical reference values, though lower than post-mortem serum concentration. Insulin concentrations in vitreous (11.50 mU/L) and cerebrospinal fluid (17.30 mU/L) were lower than blood clinical reference values. Vitreous insulin concentrations in non-diabetic control cases were lower than the estimated detection limit of the method. These preliminary results tend to confirm the usefulness of insulin determination in vitreous humour in situations of suspected fatal insulin administration. Additional findings pertaining to insulin determination in bile, pericardial, and cerebrospinal fluid would suggest that analysis performed in post-mortem serum and injection sites could be complemented, in individual cases, by investigations carried out in alternative biological fluids. Lastly, these results would indicate that analysis with chemiluminescence enzyme immunoassay may provide suitable data, similar to analysis with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immunoradiometric assay, to support the hypothesis of insulin overdose.