Preliminary Metabolomic Profiling of the Vitreous Humor from Hypothermia Fatalities.

The postmortem diagnosis of hypothermia fatalities is often complex due to the absence of pathognomonic lesions and biomarkers. In this study, potential novel biomarkers of hypothermia fatalities were searched in the vitreous humor of known cases of hypothermia fatalities (n = 20) compared to control cases (n = 16), using a targeted metabolomics approach allowing quantitative detection of 188 metabolites. A robust discriminant model with good predictivity was obtained with the supervised OPLS-DA multivariate analysis, showing a distinct separation between the hypothermia and control groups. This signature was characterized by the decreased concentrations of five metabolites (methionine sulfoxide, tryptophan, phenylalanine, alanine, and ornithine) and the increased concentration of 28 metabolites (21 phosphatidylcholines, 3 sphingomyelins, spermine, citrulline, acetylcarnitine, and hydroxybutyrylcarnitine) in hypothermia fatalities compared to controls. The signature shows similarities with already identified features in serum such as the altered concentrations of tryptophan, acylcarnitines, and unsaturated phosphatidylcholines, revealing a highly significant increased activity of methionine sulfoxide reductase, attested by a low methionine sulfoxide-to-methionine ratio. Our results show a preliminary metabolomics signature of hypothermia fatalities in the vitreous humor, highlighting an increased methionine sulfoxide reductase activity.

Levels of haemolysis have no effect on femoral vein post-mortem tryptase levels.

Haemolysis is reported to be an artefact that may alter post-mortem tryptase levels. However, previous studies did not sample peripheral blood using newly standardised methods. Recent studies have shown that some previously recognised peri- and post-mortem confounders can be muted by careful sample collection with first clamping and then sampling the femoral vein. This prospective study investigated the relationship between the degree of haemolysis of the blood samples and femoral vein post-mortem tryptase levels when sampled using this recommended method. Seventy consecutive post-mortem tryptase levels in non-anaphylactic deaths were compared to the degree of haemolysis of these samples, and results showed no significant correlation between them. The mean post-mortem tryptase level was 9.5 µg/L. This study demonstrated that the effects of haemolysis on femoral vein post-mortem tryptase was negligible when the blood was sampled using the recommended sampling method. Future studies on post-mortem tryptase as well as other typically used blood markers in forensics are recommended to adopt this method of blood sampling in routine practice.

Medicolegal Implications of Biphasic Anaphylaxis.

ABSTRACT: Biphasic anaphylaxis is an uncommon IgE-mediated condition whose pathophysiological mechanisms, risk factors, and predictive signs are not properly understood. Fortunately, the lethality of biphasic anaphylaxis, although probably underestimated, is low. Preventive clinical measures for biphasic anaphylaxis are neither standardized nor commonly applied. Furthermore, there are no laboratory protocols or anaphylactic markers to help identify the onset of biphasic anaphylaxis in clinical settings. The aim of this review is to highlight the medicolegal difficulties facing coroners and forensic pathologists in terms of the diagnosis and assessment of harm for victims and survivors of biphasic anaphylaxis.

Differences Between Central and Peripheral Postmortem Tryptase Levels.

ABSTRACT: Postmortem tryptase is a commonly used biochemical test to aid in the diagnosis of fatal anaphylaxis, which is currently recommended to be sampled from peripheral (femoral) veins because of a research showing comparatively elevated levels from central blood sources. Previous studies have used nonstandardized or nondocumented sampling methods; however, more recent research demonstrates that tryptase levels may vary depending on the sampling method. This study used the recommended sampling method of aspirating the femoral vein after clamping and compared in a pairwise comparison with aspiration of central venous and arterial blood sources (inferior vena cava and aorta) in 2 groups of 25 nonanaphylactic deaths. We found no statistically significant differences in postmortem tryptase between central and femoral vein blood; however, sporadic outliers in central blood (particularly aortic blood reaching levels above documented cutoffs for fatal anaphylaxis) were observed. Our findings provide evidence for the existing recommendations that femoral vein blood remains the preferred sample for postmortem tryptase over central blood.

Post mortem tryptase: A review of literature on its use, sampling and interpretation in the investigation of fatal anaphylaxis.

Post mortem tryptase is a commonly-used ancillary test in the investigation of possible anaphylactic deaths. Ante mortem tryptase interpretation differs from post mortem interpretation due to differing priorities, biochemical behaviours and capacity for follow-up. Additionally, post mortem tryptase sampling site, method and even cut-off levels are not standardised between facilities. This review of the literature investigates the existing research and recommendations on the use of post mortem tryptase in suspected anaphylactic deaths. Currently, autopsy recommendations suggest early sampling, standardised sampling technique with clamping of and aspiration from the femoral vein, and for the results to be interpreted within the wider autopsy and clinical context. Areas in need of further research include the effects of cytolysis on tryptase levels and studies to stratify differing tryptase levels based on type of death and anaphylactic trigger.

Combining Postmortem Cerebrospinal Fluid Biochemistry With Lung-to-Body Ratio to Aid the Diagnosis of Salt Water Drowning.

Diagnosing drowning as a cause of death can pose many challenges for the forensic pathologist and a number of ancillary tests have been proposed to assist in the diagnosis, whether the body was in salt water or fresh water. Although elevated vitreous humor sodium and chloride is a reliable marker, its limitation to prolonged immersion has resulted in the recent investigation of cerebrospinal fluid (CSF) sodium and chloride as alternative matrix in cases of longer or unknown immersion times. This study investigated postmortem CSF from lumbar puncture (CSF_L_Na_Cl) and ventricular aspiration (CSF_Vent_Na_Cl), as well as lung/body (LB) ratio in the diagnosis of salt water drowning and performed comparison and combination testing of methods to improve diagnostic accuracy of the drowning diagnosis. This study found that CSF_L_Na_Cl was the most accurate method (89%) in the given cohort, but that CSF_Vent_Na_Cl and LB combined was the second most accurate method (83%), exceeding CSF_Vent_Na_Cl (77%) and LB (81%) used alone. These findings are useful for stratifying and prioritizing postmortem samples in the investigation of salt water drowning and also have significance for future studies using this methodology to combine and compare the accuracy of different investigations.

Postmortem Tryptase Level in 120 Consecutive Nonanaphylactic Deaths: Establishing a Reference Range as <23 µg/L.

Postmortem tryptase is a useful biochemical test to aid the diagnosis of anaphylaxis. Multiple perimortem and postmortem factors have been documented to cause an elevation in postmortem tryptase level. One factor that was recently recognized to have an impact on postmortem tryptase level is correct sampling technique. A recent study recommended aspirating blood samples from a clamped femoral/external iliac vein to be used for reliable postmortem tryptase analysis. This study sampled 120 consecutive nonanaphylactic deaths in which all the peripheral bloods were sampled as recommended. Postmortem interval, resuscitation, different nonanaphylactic causes of death, sex, and age did not show any statistical significant relation to postmortem tryptase level in Student t test, Pearson correlation, and univariate and multivariate analyses. The mean (SD) postmortem tryptase level was 8.4 (5.2) µg/L (minimum, 1.0 µg/L; maximum, 36.1 µg/L; median, 7.3 µg/L). Using nonparametric methods, the postmortem tryptase reference range in nonanaphylactic death was established as <23 µg/L (97.5th percentile).

Elevation of Postmortem Cerebrospinal Fluid Sodium and Chloride Levels Is a Potential Adjunct Test in the Diagnosis of Salt Water Drowning.

Postmortem vitreous humor biochemistry is a useful test in the diagnosis of salt water drowning (SWD). A significant limitation of vitreous humor is the potential effect of prolonged immersion. A recent animal study and case report suggested that cerebrospinal fluid biochemistry may be an alternative to vitreous because it is more resistant to the effects of immersion, given its protected anatomical location. This study compared postmortem cerebrospinal fluid sodium and chloride (PMCSC) levels collected via ventricular aspiration (PMCSC_V) and via lumbar puncture (PMCSC_L) in 13 SWD and 31 nonimmersion deaths. It showed a significant elevation in PMCSC levels in SWD deaths for both PMCSC_V and PMCSC_L (P < 0.05). The areas under the curve on the receiver operating characteristic curves for PMCSC_V and PMCSC_L were 0.73 and 0.83, respectively. The optimal cutoff for PMCSC_V was 216 mmol/L (sensitivity, 0.60; specificity, 0.72; likelihood ratio, 1.80; positive predictive value, 0.45) and for PMCSC_L was 241 mmol/L (sensitivity, 0.78; specificity, 0.73; likelihood ratio, 2.89; positive predictive value, 0.46). This study supports PMCSC levels as another biochemical test that can potentially aid in the diagnosis of SWD, particularly in cases where vitreous humor samples are unavailable or uninterpretable.

Biomarkers of Cerebral Damage in Fatal Hypothermia: Preliminary Results.

The identification of hypothermia as the cause of death remains challenging in forensic pathology because of unspecific radiological, morphological, and biochemical results. Hyperemia, edema, and petechial hemorrhages within the cerebral parenchyma were described in cases of death by hypothermia. On the other hand, the effect of low temperatures in the brain has been speculated to cause local injuries on a cellular level with potential occurrences of necrosis and inflammation. In the study herein described, endocan, alkaline phosphatase, neuron-specific enolase, S100 protein subunit B, glial fibrillary acidic protein, and C-reactive protein were measured in postmortem serum from femoral blood and cerebrospinal fluid in a series of hypothermia fatalities and control cases. The combination of data collected failed to identify a specific biochemical profile for death by hypothermia in postmortem serum and/or the cerebrospinal fluid, thus suggesting that an alternative panel of brain damage biomarkers indicative of diffuse hypoxic brain injury needs to be defined in hypothermia fatalities.

The forensic spleen: Morphological, radiological, and toxicological investigations.

The spleen is only uncommonly investigated in the forensic setting. Thorough examinations are performed in some specific situations such as splenic trauma (including iatrogenic trauma from cardiopulmonary resuscitation attempts), anaphylaxis, drowning, and sepsis-related deaths. The aim of this review is to present the available literature focusing on a few selected splenic diseases as well as forensic investigations performed on the spleen in order to summarize the most frequent situations in which this routinely unexplored organ may merit more extensive examination.

Biochemical investigations performed in pericardial fluid in forensic cases that underwent postmortem angiography.

Postmortem biochemical investigations in vitreous humor samples collected before and after performing multiphase postmortem computed tomography angiography were performed in the past and demonstrated that specific contrast material injection allowed perfusion and radiological identification of the main vessels of the eye to be obtained without any changes in vitreous humor composition. In the study presented herein, we aimed to test whether the injection of the same contrast material using the same postmortem angiography protocol might influence pericardial fluid composition. Postmortem biochemical investigations were performed on pericardial fluid samples collected from bodies that underwent postmortem angiography (n = 16) prior to and post angiography. Two pericardial fluid samples were analyzed. No statistically significant differences were noticed among levels of any tested markers (urea nitrogen, creatinine, uric acid, C-reactive protein, procalcitonin, beta-hydroxybutyrate, and total IgE levels) in pericardial fluid samples collected prior to and post angiography, leading to the conclusion that pericardial fluid sampling can be delayed until after postmortem angiography when a specific contrast material injection is used.

Elevated Cerebrospinal Fluid Sodium and Chloride Levels in a Saltwater Drowning Death.

To ascribe a cause of death from drowning in a body immersed in water can be difficult because of the absence of specific postmortem findings and unreliable ancillary tests. Postmortem vitreous biochemical analysis is documented to be a useful adjunct ancillary test to aid the diagnosis of saltwater drowning. A major confounding factor in using postmortem vitreous is the effect of electrolyte diffusion and water osmosis during immersion. A recent animal study suggested that cerebrospinal fluid (CSF) biochemical analysis, which is unaffected by immersion, may be an alternative. However, to date, there are no human data to support this. We report a saltwater drowning death from presumed suicide in which the postmortem CSF sodium and chloride level was elevated compared with nonimmersion deaths. This case gives evidence to support the potential use of postmortem CSF sodium and chloride level as an adjunct to the diagnosis of saltwater drowning.

Postmortem IgE determination in coronary artery disease.

Allergic, IgE-mediated inflammation is thought to play a role in atherogenesis and atherosclerotic disease progression. In this study, total IgE and mast cell tryptase were measured in a series of forensic autopsy cases including non-allergic cardiac deaths (14 cases with minimal or no coronary atherosclerosis, 14 cases with significant coronary artery atherosclerosis without acute coronary thrombosis, and 14 cases with significant coronary artery atherosclerosis and acute coronary thrombosis or myocardial infarction) and non-allergic non-cardiac deaths (21 cases with death due to hanging and 21 cases with death due to intracranial gunshot wounds), in order to correlate laboratory results with morphological findings and compare them to conclusions reported in the clinical setting. In cardiac death cases, postmortem serum total IgE levels were increased in 7 out of 42 cases and mast cell tryptase levels were increased in 3 out of 42 cases. In non-cardiac death cases, postmortem serum total IgE levels were not increased in 39 out of 42 cases and mast cell tryptase levels were not increased in any of these cases. These preliminary findings seem to indicate that a portion of coronary deaths characterized by coronary artery atherosclerosis of various severities are also characterized by increased total IgE and mast cell tryptase levels, thus corroborating the data previously reported in both clinical and forensic literature on this topic as well as the necessity of combining morphological investigations focusing on the heart and coronary arteries with biochemical analyses.

A serum metabolomics signature of hypothermia fatalities involving arginase activity, tryptophan content, and phosphatidylcholine saturation.

INTRODUCTION: Hypothermia is a potentially lethal condition whose postmortem diagnosis is often complex to perform due to the absence of pathognomonic lesions and biomarkers. Our first study of human serum and urinary metabolome in hypothermia fatalities sought novel biomarkers with better diagnostic performances than those already existing. MATERIAL AND METHOD: Thirty-two cases of hypothermia deaths and 16 cases excluding known antemortem exposure to cold or postmortem elements suggesting hypothermia were selected. A targeted metabolomic study allowing the detection and quantitation of 188 metabolites was performed on collected serum and urine using direct flow injection (FIA) and liquid chromatography (LC) separation, both coupled to tandem mass spectrometry (MS/MS). Amino acid quantification was also carried on using an in-house LC-MS/MS method in order to replicate the results obtained with the metabolomic study. RESULTS: A discriminant metabolic signature allowing a clear separation between hypothermia and control groups was obtained in the serum. This signature was characterized by increased arginase activity and fatty acid unsaturation along with decreased levels of tryptophan in hypothermia fatalities compared to controls. By contrast, no discriminant metabolic signature separating hypothermia from control fatalities was found in urines. DISCUSSION: The serum metabolic signature of hypothermia fatalities herein observed pointed toward metabolic adaptations that likely aimed at heat production enhancement, endothelial function, and cell membrane fluidity preservation. Novel biomarkers potentially useful in a hypothermia diagnosis were also identified.

Biochemical Differences Between Vitreous Humor and Cerebral Spinal Fluid in a Death From Diabetic Ketoacidosis.

Biochemical analysis of cerebrospinal fluid (CSF) and vitreous humor (VH) forms an important diagnostic ancillary test at autopsy. Cerebrospinal fluid can be sampled from the ventricular system (VA) and from lumbar puncture (LP), whereas VH can be sampled from the orbits. Biochemical electrolytes seem to vary between VH and CSF collected from different sites, but whether there is any difference in glucose and ß-hydroxybutyrate is unclear. We present a case report of a 21-year-old man who died of diabetic ketoacidosis confirmed at autopsy. Ventricular system, LP, and VH were biochemically analyzed and showed that glucose was highest in VH (41 mmol/L) and was 6 and 8 mmol/L higher than CSF in the LP and VA, respectively. ß-Hydroxybutyrate was also highest in VH (20 mmol/L) and was 5 and 6 mmol/L higher than LP and VA, respectively. Our findings suggest that postmortem CSF glucose and ß-hydroxybutyrate may not truly reflect that of VH and vary between CSF sampling sites.

The forensic spleen: Morphological, radiological, and toxicological investigations.

The spleen is infrequently investigated in forensic pathology routine. Thorough examinations are performed in very specific situations such as splenic trauma (including iatrogenic trauma from cardiopulmonary resuscitation attempts), anaphylaxis-related deaths, drowning and sepsis. The purpose of this review article is to provide a general overview of available literature focusing on a few selected splenic diseases as well as available forensic investigation techniques performed on the spleen in order to summarize the most frequent situations of forensic interest in which this routinely unexplored organ may merit more extensive examination.

Appearance/Image- and Performance-Enhancing Drug Users: A Forensic Approach.

Image- and performance-enhancing drugs include a wide range of substances used to promote physical changes to enhance appearance. Anabolic androgen steroids are the most widely used image- and performance-enhancing drugs along with a wide variety of additional substances. The aim of this study was to identify the pathological changes pertaining to the cardiovascular system possibly involved in the death and characterize the substances associated with steroid use and their possible role in the death. A series of deaths involving image- and performance-enhancing drug users were selected. Nandrolone and testosterone were the most frequently identified steroids. The most commonly illegal drugs found were tetrahydrocannabinol, cocaine, and methadone. Antidepressants, opioids, benzodiazepines, and barbiturates were also found. These findings highlight that results obtained from postmortem investigations should be meticulously evaluated to understand the weight that each identified substance may have played in the death process and exclude any alternative causes of death.

Differences in Sampling Site on Postmortem Cerebrospinal Fluid Biochemistry: A Preliminary Study.

Cerebrospinal fluid (CSF) is often analyzed at postmortem. The presented preliminary study compared postmortem CSF samples for biochemical analysis from the subarachnoid space around the spinal cord and ventricular space of the brain. This study compared 15 paired CSF samples in which the CSF from the subarachnoid space via lumbar puncture had higher sodium and chloride levels and lower magnesium and potassium levels than CSF from the ventricles. The differences correlated significantly with the deceased's age and had a similar trend with postmortem interval. This study suggests that CSF from different collection sites has different electrolyte concentrations, which are age and possibly postmortem interval dependent. When collecting CSF, the pathologist should document the collection site, age, and postmortem interval, and the mixing of CSF samples from different sites should be avoided. Further studies are warranted to clarify other possible reasons to explain the observed differences.