Death in Epilepsy: Ictogenic Vs. Cardiogenic Mechanisms and Immunoassays in Postmortem Interval Estimation – Case Report

Abstract

Background

Epilepsy may cause sudden death. More scenarios are possible: sudden unexpected death in epilepsy (SUDEP), cardiac and non-cardiac mechanisms of death. This study presents the case history of a 57-year-old person known for epilepsy who experienced a final tonic-clonic seizures with ictal cardiorespiratory arrest shortly after hospital admission. The present study aimed to determine 1) the cause of death and possible mechanisms; and 2) reference values for immunoassays at 36.8-hour postmortem interval.

Materials and methods

A full medico-legal autopsy was performed. Postmortem findings and a large panel of investigations are provided: anatomic pathology, pericardial chemistry investigations, forensic serology and toxicology. Immunoassays including sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) with Western blotting for vinculin and enzyme-linked immunosorbent for cardiac TnT, vinculin and desmin were used to assess the postmortem interval.

Results

Cause of death, mechanisms and medico-legal causality are debated. Autopsy findings and relevant investigations did not reveal a single identifiable cause of death incompatible with life. Even more, SUDEP appears improbable and status epilepticus not documented. An ictogenic cause of death with autogenous and cardiac mechanisms involvement, enhanced by concomitant conditioning factors, a severe metabolic acidosis, respiratory failure with impaired ventilation and extensive myocardial fibrosis appears more probable. SDS-PAGE with Western Blot was able to detect the native molecular band 117 kD of vinculin and three molecular products of degradation at 90 kD, 87 kD, 84 kD at 36,8-hour postmortem interval and therefore confirmed the postmortem interval, while ELISA detected cardiac TnT (178.85 pg/mL), vinculin (570.96 pg/mL) and desmin (565.77 pg/mL) in the skeletal muscle.

Conclusions

This case highlights the important role played by medico-legal autopsy in death investigation in epilepsy. If for legal purpose causality is required, causes of death and conditioning factors are to be extensively investigated and connected. ELISA delivered benchmarks references at 36.8-hour postmortem interval and provided to be a valuable immunoassay method in estimation of postmortem interval.

INTRODUCTION

Epilepsy may cause either violent (e.g., traumatic, intoxication, etc) or non-violent causes of death. Epilepsy-related deaths are attributed to: a) epilepsy such as status epilepticus, sudden unexpected death in epilepsy (SUDEP), ictal syncope; b) cardiac mechanisms of death such as "epileptic heart", sudden cardiac death (SCD), sudden unexpected cardiac death (SUCD), unexplained death, cardiogenic syncope, commonly associated with postictal cardiorespiratory dysfunction due to hypoxia, CO2 retention, hypoventilation, bradycardia, asystole and ventricular arrhythmias; c) non-cardiac mechanisms of death such as (3a) brain causes such as night apnea (1), autonomic nervous dysfunctions (2), deregulation of the central control of autonomic functions made possible under catecholamine influx with subsequent hypoxemia on heart (3), brainstem mechanisms (4), corticolimbic circuit deficiency (5), (3b) other non-cardiac causes such as metabolic state disturbances [metabolic acidosis with normal and high anion gap groups may precipitate epilepsy and seizures as seizures may generate metabolic acidosis (6, 7)]; and d) violent death, e.g., trauma with injuries by fall or intoxication (antiepileptic drug use, drug adverse reactions, overdose (8).

Sudden unexpected death in epilepsy (SUDEP), as a non-traumatic, non-violent and non-drowning fatality, usually follows a generalized tonic-clonic seizure (GTCS), or without a seizure (4) that follows early postictal [initially central respiratory dysfunction progressing to a terminal apnea, later followed by cardiac arrest (9)], or after a short period of partly restored cardiorespiratory function (with apnea and cardiac arrest (4) soon after the onset of apnea) in all deceased patients (MORTEMUS study) (10).

Sudden unexpected death in epilepsy is generally considered a result of seizure-related cardiac dysfunction, respiratory depression, autonomic nervous dysfunction, or brain dysfunction (11) and has an incidence of about 1.2 per 1 000 person-years in the general epilepsy population (12). Nevertheless, SUDEP excludes documented status epilepticus (4, 13). The unified classification of near, possible, probably and definite SUDEP and SUDEP plus conditions/comorbidities is very useful and helps in understanding the pathophysiology and discriminating among cases (13) (Figure 1).

We found a relevant case to illustrate the above-mentioned scientific debates regarding the cause and mechanisms of death in epilepsy. The objectives of this case study overlap prosecutor requests: the cause of death, medico-legal causality and a validation of postmortem interval (36.8 hours) which come out from hospital data (see Figure 2).

CASE DESCRIPTION

An unidentified 57-year-old homeless male with a history of alcohol consumption and chronic epilepsy and multiple admissions for generalized tonic-clonic seizures (GTCS) presents to the emergency department of the hospital. He refuses hospitalization and receives treatment as usual. The next day, at 8:20 pm, he presents again at the same hospital, probably post-ictal, with weakness and confusion. Clinical examination at 8:25 pm reveals dizziness, asthenia, Glasgow Coma Scale, GCS 15, heart rate (HR) 55 and respiratory rate (RR) 5 (a severe association of bradycardia and bradypnea that orientated the healthcare for an emergency case). At 8:26 pm, electrocardiogram (ECG) and arterial blood gas (ABG) are urgently requested and recorded; the ECG graphical representation is not available at the patient file. After ABG sampling (approximately 8:26-8:30 pm), lying on the bed for clinical examination the patient experienced a generalized tonic-clonic seizure, GTCS: shortly after the GTCS onset at 8:30 pm (10 minutes after admission) presents ictal cardiorespiratory arrest. Cardiorespiratory resuscitation is applied, but cardiac and pulmonary functions are irresuscitable and irreversible ceased and death is pronounced at 8:46 pm, 26 minutes after admission. The very rapid development of clinical symptoms, unexpected cardiorespiratory arrest and death pronouncement after only 26 minutes from hospital admission determine a full medico-legal autopsy, officially ordered for body identification, health-care management investigation, cause of death assessment, medico-legal causality and postmortem interval validation. Autopsy is performed the next day at 9:30 am.

FIGURE 1.

Infographic: a suggestive diagram of the definition and classification of SUDEP and non-SUDEP cases (adapted from Box 3 of "Unifying the Definitions of Sudden Unexpected Death in Epilepsy") (13) (original creation using Mind the Graph)

FIGURE 2.

Graphical abstract, depicting objectives of the study, methodology, possible cause of death and mechanisms, value references at 36.8-hour postmortem interval (PMI) using two immunoassay methods, including SDS-PAGE with Western Blot and ELISA (original creation using Mind the Graph).

METHODS

After death, the body was kept at the hospital morgue, cooling at ambient temperature until the morning and then refrigerated at 4°C. The refrigeration temperature was constantly 4°C in the hospital and legal medicine institute morgue. Autopsy room at temperature around 20°C. Postmortem interval was initially calculated from hospital documents. The calculated postmortem interval (PMIc) = date and time of autopsy (24.10.2024, 9:30 am) – date and time of death (22.10.2024, 8:46 pm) = 36,8-hours (the calculation includes for minutes, 0.8 hours, the corresponding fraction from one hour).

Blood was sampled for identification (blood group) and postmortem toxicology (to exclude a violent death by drugs intoxication or other substances). Pericardial fluid was sampled for thanatochemistry: creatinine kinase-MB, troponin I (cTnI) and myoglobin. For anatomical pathology, organ samples were preserved on buffered formalin solution 10% (from standard 40% formalin, v/v, the resulting liquid solution containing 4% formaldehyde) and kept for preserving and fixation of the tissue samples at least 24 hours (2 mm/hour formalin tissue penetration rate). Histopathology technique was applied after fixation for paraffin embedding, microtome sections and H&E staining.

For PMI estimate we selected available immunoassay methods, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blotting for vinculin and enzyme-linked immunosorbent assay (ELISA) for cardiac troponin T (cTnT), vinculin and desmin. Sampling from the skeletal muscle (M. vastus lateralis – quadriceps), 5/5/5 cm at the beginning of the autopsy, was prepared and immediately sent on ice to the serology laboratory to be prepared for protein extraction on the same day following the following steps: 1) removing of fat, connective tissue by dissection and washing with ice-cold phosphate-buffered saline (PBS); 2) sampling on ice in smaller fragments 0.1 g fragments, washed three times with cold PBS (4°C) to remove any blood residues; 3) dissecting into even smaller pieces, equal in size and shape to obtain a larger contact surface with the extraction buffer; 4) transfer on ice, into a 2 mL tube containing 1 mL of extraction buffer (radioimmunoprecipitation assay buffer and 10 μL of protease inhibitor cocktail incubated at room temperature 22°C for 30 minutes; and 5) homogenization, centrifugation with the supernatant transferred on ice to a 1.5 mL tube and frozen at -80°C until further use.

For protein concentration determination, we used the Pierce BCA Assay Kit from Thermo Scientific, which contained reagents for the standard curve at the following concentrations: 0.125; 0.25; 0.5; 0.75; 1; 1.5; 2 mg/mL. For SDS-PAGE, 30 μg of protein denatured at 95°C for five minutes were used. For electrophoresis in running buffer (Tris/glycine/SDS), Mini Protean TGX Stain-Free Protein Gels from Bio-Rad were used. Transfer conditions comprised constant voltage of 150 V for approximately 75 minutes. Electrophoresis gels were transferred to blotting membranes for immunodetection using Trans-Blot Turbo Transfer System, polyvinylidene fluoride (PVDF) 0.2 µm membrane and transfer conditions including 2.5 A and 25 V for seven minutes. Membranes blocked in blocking buffer and incubated in primary antibodies – Mouse Anti Vinculin (VMA00895 Bio-Rad) diluted in blocking buffer 1:1 000 and secondary antibodies – goat anti mouse (STAR207P) diluted in blocking buffer 1:3 000. After each incubation step, washes were performed with TBST (Tris buffered saline with Tween® 20) (commercial name for Polysorbate 20). Antibody binding was visualized by adding chemiluminescent substrate (Clarity Western ECL Substrate – Bio-Rad) and documented using a digital imaging system (ChemiLITE – Cleaver Scientific). Gel analysis was performed using Cleaver geneQUANT software version 4.3.9.0.

For ELISA, a semi-automatic sandwich enzyme immunoassay equipment is available. We did not perform dilutions. We used troponin T (ELK3961 – human cTnT/TNNT2, troponin T type 2, cardiac); vinculin (ELK2218 – human vinculin ELISA kit) and desmin (ELK2654 – human desmin ELISA kit). Each kit had a standard curve which was obtained by gradually dilution series of standard working solution according to kit instructions. After equilibrating the kit to room temperature, 100 μL of each standard and sample were pipetted and incubated at 37°C for 80 minutes. The liquid in the plate was discarded, then 200 μL 1× wash buffer were added to each well and the plate was washed three times. After patting and drying with clean absorbent paper, 100 μL of biotinylated antibody working solution (1×) were added to each well. Incubation at 37°C for 50 minutes was done. This was repeated with 100 μL 1× streptavidin-HRP working solution, incubated at 37°C for 50 minutes and again with 90 μL TMB substrate solution incubated at 37°C for 20 minutes in the dark. Finally, we added 50 μL Stop Solution to each well and shaked the plate on a plate shaker for one minute to mix. The optical density is measured at 450 nm (the measure of the absorbance of light). Results are calculated with Magellan software (Bio-Rad) based on the standard curve concentrations.

RESULTS

Autopsy findings

Postmortem findings are nonspecific: brain with moderate edema, heart enlargement, pulmonary edema with rare venous thrombosis, liver steatosis, chronic pancreatitis with cytosteatonecrosis, chronic pyelonephritis with distorted medulla, autolysis.

Anatomical pathology

Samples of anatomical pathology are illustrated in Figure 3.

Other investigations: forensic serology, forensic toxicology, pericardial fluid thanatochemistry and immunoassays methods

Blood group A (D) Rh positive for identification. Postmortem toxicology excludes a violent death (blood alcohol 0 g‰, urine alcohol 0.05 g‰, anti-convulsant drugs absent). Pericardial fluid thanatochemistry exhibit creatinine kinase-MB 2368.3 U/L, troponin I (cTnI) positive, myoglobin positive. SDS-PAGE and Western Blotting detect vinculin products degradation at 90 kD, 87kD and 84 kD band. ELISA succeeded to detect cardiac troponin T, cTnT, 178.85 pg/mL, vinculin 570,96 pg/mL and desmin 565.77 pg/mL.

FIGURE 3.

Anatomical pathology: (a) brain – brain edema, perivascular micro hemorrhages (H&E x 20); (b) heart – extensive myocardial fibrosis dissecting the cardiac tissue (H&E X 20); (c) C9 slightly positive in heart sample (H&E x 200); (d) lung – pulmonary edema: lung alveoli spaces are filled with eosinophilic fluid (H&E X 20); (e) lung – scattered pulmonary vessel thrombosis: in the picture, the thrombus largely occupies the vessel and adheres to the wall (H&E x 20); (f) liver steatosis – large lipid droplets accumulate in hepatocytes without inflammation (H&E x 20); (g) pancreas – pancreatic cytosteatonecrosis with lobular pancreatic panniculitis and adipocyte necrosis ("ghost cells" appearance) without hemorrhages (H&E x 20); (h) kidney – chronic distorted renal parenchyma, tubule epithelium desquamation and detachment (center-right), luminal casts, suggestive of autolysis. The histopathological features of autolysis are difficult to differentiate to acute tubular necrosis features, but key elements of renal tubular necrosis are missing cells vacuolization, tubular epithelial whorls and hematopoietic cells in vasa recta), (H&E x 20).

DISCUSSION

Accumulated data suggests that ictal cardiac dysfunction plays a limited role in SUDEP and should be distinguished from post-ictal cardiac rhythm disorders (i.e., bradycardia, asystole, or ventricular fibrillation) that usually follow central peri-ictal respiratory disorders (hypoxemia) (14). Nevertheless, ictal and postictal apnea, hypoventilation and hypoxia in their turn increase the risk of SUDEP (15). In long term epilepsy, as in the case described here, it may be difficult to distinguish ictal asystole syncope from cardiac asystole syncope as well ictogenic v. cardiogenic cause of death, commonly intricated in the so-called "epileptic heart" (16). The "epileptic heart" may develop heart failure, arrhythmias and reduced heart rate variability (15) and precipitate SCD risk, separately from SUDEP. This could be the case in our report, but because the patient did not have a documented medical history, it is only supposably. In chronic epilepsy, the heart is structurally altered ("epileptic heart") (16) at the myocardium level, cardiac conduction system and coronary vasculature because of subsequent rises in catecholamine levels and repetitive episodes of hypoxemia during seizures (i.e., extensive myocardial fibrosis, see Figure 3b, 3c).

International League Against Epilepsy (ILAE) definition expresses that cardiac comorbidities should not be considered a known cause of death (15). Comorbidities are conditions in the causality link of events and the cause of death is still different (Figure 4).

In acute clinical epilepsy, seizures may induce ictal tachycardia, bradycardia and asystole as autonomic responses. In chronic clinical epilepsy, attenuated and variable arrhythmia complications are frequently found (prolonged QTc intervals, elevated T waves, increased P-wave) (16, 17). In some chronic epilepsy cases, seizures induce cardiac stress linked to the chronic release of catecholamines (15) that may mimic an acute ischemic heart disease, like in Takotsubo syndrome, or generate a simultaneity of cardiac and neurological conditional factors (18). This condition must be distinguished from SUDEP and SCD (sudden unexpected death by cardiac arrest due to cardiac causes within an hour from the symptoms onset or without symptoms with or without known causes), sudden arrhythmic death syndrome (SUDS) or unexplained death (15, 17). Cardiogenic syncope in epilepsy is associated with arrhythmias, structural heart disease, or autonomic dysfunction; ictogenic syncope results from seizure-induced cerebral hypoperfusion but discrimination is often challenging (19). In the case presented by us, we appreciated that the syncope was cardiogenic. Although ECG may objectify the cardiac status, this was not available in our case.

Ictal cardiac arrhythmias as manifestations of epileptic seizures use common autonomic pathways, probably initiated from the amygdala, gyrus cinguli, insular cortex, frontopolar, or frontal-orbital regions and consist of supraventricular tachycardia, sinus tachycardia, sinus bradycardia, sinus arrest, atrioventricular block and asystole (20, 21).

Cause of death, mechanisms

Arterial blood gas, ABG and data are deviated from normal in the case presented and sustain a metabolic disorder. A three-step method was used for the diagnosis of the metabolic disorder (7): 1) anion gap determination: applied formula: Anion gap = Na-Bicarb-Cl = 144 mEq/L – 10.1 mEq/L – 113 mEq/L = 20.9 mM (normal <12 mM); diagnosis anion-gap metabolic acidosis (AGMA); 2) bicarbonate determination (10.1 mM, normal <22 mM) low bicarbonate; 3) "delta delta" (applied formulas: ∆ Anion gap = (Anion gap) – 10 = 20.9 mM – 10 = 10.9 mM and ∆ Bicarbonate = 24 – Bicarbonate = 24 – 10.1 = 13.9; diagnosis non-anion-gap metabolic acidosis (NAGMA). Final diagnosis is AGMA + NAGMA, non-compensated (RR 5/min), respiratory failure Horowitz 1 (Horowitz index P (PaO2) / F (FiO2) = 59 mm Hg / 0,40 (5 L/min nasal canula is 40% O2) = 58,6 (<100 denotes severe lung ventilation impairment).

In this case, an intricate pathology accumulates to produce this severe metabolic disorder: lung (pulmonary edema, thrombosis, respiratory failure), kidney (chronic pyelonephritis), pancreas (citosteatonecrosis) (22), liver (liver steatosis), toxic (alcohol consumption antecedents), metabolic (glycemia 178 mg/dL), skeletal muscle (seizures). Then again metabolic disorder induces acidosis, hypoxemia, enhancing hypoxemia induced by respiratory failure and ventilation impairment (PaO2 59 mm Hg). Generalized tonic-clonic seizure, GTCS that follow deepens even more hypoxemia that triggers dysrhythmia in a most probably "epileptic heart" (i.e., extensive myocardial fibrosis, see Figure 3b, c) to produce cardiorespiratory arrest.

Postmortem findings did not reveal any incompatible with life cause of death, violent or non-violent (trauma-related, toxicological, etc). Some of the anatomical pathology findings correlates postmortem findings (e.g., cerebral edema, pulmonary edema, liver steatosis, pancreatic cytosteatonecrosis), other support new findings such as the extensive myocardial fibrosis. The present case is not SUDEP or near SUDEP either SCD or SUDS because there are known conditions as comorbidities that clearly manifest (e.g., myocardial fibrosis, metabolic acidosis, etc).

Myocardial ischemia may be induced by seizures, especially in patients with cardiovascular risk factors (23). Postmortem cardiac markers from the pericardial fluid are particularly useful when the cause of death is inconclusive (4, 24). Less than 30% of patients with GTCS have elevated troponin levels, which normally sustain transient myocardial hypoxemia (25); the case we present exhibits cardiac markers in pericardial fluid that gives support to cardiac hypoxemia: troponin I (cTnI) (19), CK-MB 2368.3 U/L above normal (105-154 U/L) (26), positive values of myoglobin (26) and C9 (27) (see also Figure 3 B).

Tachycardia is the most common ictal cardiac manifestation (3), similar in GTCS and focal seizures, while seizures with bradycardia with ictal asystole are rarer (28). After a generalized seizure, there is a greater risk of asystole in patients with severe post-ictal apnea and cardiac arrhythmias (10). An ECG again would have been very useful.

The patient would benefit from a prior healthcare under medical supervision with cardiac assessment and ECG monitoring to lower the risks for ictal/post-ictal death and SUDEP (29).

Limitations of cause of death and mechanisms study

In this case, the limitations in cause of death include undocumented cardiac antecedents and unavailable ECG (which was performed, but its graphical expression was missing from the patient's file). The ECG may discriminate between ictal and post-ictal patterns of cardiac arrhythmias, e.g., tachycardia, asystole, bradycardia and atrioventricular conduction block (14) and helps to ascertain ictogenic vs. cardiogenic mechanisms of death as well as the rhythm involved, tachycardia with final fibrillation or asystole. Ictal asystole syncope may be difficult to distinguish from cardiac asystole syncope.

Medico-legal causality

It implies the medico-legal assessment of events with juridical implications to be used in a court of law. Our approach identifies two main possibilities with only one outcome: 1) a non-documented status epilepticus (in this case, at least two GTCS in two days); and 2) an ictogenic cause of death enhanced by concomitant conditioning factors, i.e., a severe metabolic acidosis, respiratory failure with impaired ventilation and extensive myocardial fibrosis.

The ictogenic cause of death with autogenous and cardiac mechanisms involvement is better documented by clinical, postmortem findings, anatomical pathology, post-mortem cardiac markers positivity and rise (CK-MB) compared to status epilepticus.

The causality sequence appears as follows (Figure 4): 1) the patient with epilepsy who was not under medical control develops heart conditions ("epileptic heart", extensive myocardial fibrosis due to chronic hypoxia); 2) cardiac conditions (hypoxia) lead to respiratory failure with ventilation impairment due to pulmonary edema; 3) non-compensated metabolic acidosis – AGMA and NON-AGMA (because of respiratory failure) – has intricate etiology and initiate 4) a generalized tonic-clonic seizure (GTSC), 5) which precipitates a cardio-respiratory arrest (CRA) (severe hypoxia, PaO2 59 mm Hg). The death is non-traumatic and non-violent (toxicology negative for drugs); health care management was according to protocols in the very narrow window of the clinical emergency management (26 minutes from hospital admission).

As officially requested, medico-legal causality as evidence is prone to assessment in the court or under judicial investigation (30, 31). Admissibility in a court of law is tested using Daubert standards (checking scientific validity, reproducibility, predictability, reliability) (32) and Frye Standard (based on the general acceptance of the evidence) (33).

FIGURE 4.

Infographic: a schematic and didactic representation of medico-legal causality as aggregated medical data suggests presenting the most important pathological events in their temporal connection. Nevertheless, practically all conditional factors combine to reinforce negative feedback (original creation using Mind the Graph).

Postmortem interval

In this case, an official investigation of health care management and a request for PMI assessment and validation offer the opportunity to study the behavior and values for several biological markers (cardiac troponin T, vinculin and desmin) at an already known PMIc of 36.8 hours (hospital data). The postmortem interval was assessed using protein degradation available immunoassays, including SDS-PAGE and Western Blotting and ELISA (34, 35), and selected biomarkers including cardiac troponin T, vinculin and desmin. Cardiac troponin T has three isoforms: cTnT, cTnI and cTnC (36). We were interested to investigate the cardiac isoform cTnT to search availability within the skeletal muscle knowing that its sources were the skeletal muscle and the heart (37). While cTnI isoform elevation appears more robustly linked to myocardial pathology (37), cTnT in skeletal muscle in post-seizure and regenerating status of the muscle after rhabdomyolysis shifts the interpretation more closely to its muscular source (38) in the absence of evidence of ischemic myocardial disease. Vinculin and desmin as cytoskeleton proteins prove reproducible and useful results when degrading in PMI estimation using immunoassays methods (39-41).

SDS-PAGE and Western Blotting we used detect the native vinculin band of 117 kDa usually depicted in reference studies with human samples in early PMI < 0.5 day (41), <24 hours PMI (39) or days 1, 2, 3 (40) and depicts also vinculin degradation bands of 84 kDa, usually depicted from day 1 and day 3 < PMI 48 hours (40), bands of 90 kD and 84 kDa, usually depicted in PMI < 72 PMI hours (30) and therefore validated the intermediate PMI (PMIc of 36.8 hours) in this case. Bands of 75 kDa and 63 kDa depicted in intermediate PMI (~2.5 PMI days) (41) were not revealed but additionally it depicted vinculin degradation product band of 87 kDa that cited studies (39-41) did not reveal.

There again, ELISA succeeded in detecting cTnT in the skeletal muscle, 178.85 pg/mL. We found only one reproducible study in the literature which had important similarities to our study to compare with (38); both studies used ELISA, a similar range of PMI (30-40 hours), sampling from skeletal muscle, muscle with regenerative conditions (epilepsy with rhabdomyolysis by generalized tonic-clonic seizure, GTCS v. polymyositis and Duchenne muscular dystrophy (38). The above-mentioned study reveals that ELISA may not find cTnT in the skeletal muscle in normal skeletal muscle (non-regenerative muscle) v. regenerative skeletal muscle (e.g., polymyositis, Duchenne muscular dystrophy, etc). We were still able to find cTnT using ELISA in skeletal muscles probably because the skeletal muscle we used as sample was a regenerative skeletal muscle in chronic epilepsy and recently antecedents of repeated GTCS. Seizures may induce rhabdomyolysis (42) (creatine kinase, a specific marker for rhabdomyolysis – however, not required).

When comparing ELISA detection limits, we found differences between our study and Bodor's report (38): 178.85 pg/mL) v. 0.8 mg/g (equivalent to 0.8 mg/mL) (38). There are several possible explanations: 1) our research benefits from a better sensitivity from recent ELISA equipment and kits; 2) the case presented by us has a different cause of death –epilepsy v. polymyositis and Duchenne muscular dystrophy (38); and 3) the temperature in our study (corporeal study) was at 4°C cadaver refrigeration temperature and 20-22°C laboratory temperature with sample preparation on ice v. extracorporeal study (38) with -80°C frozen tissues and defrosing with sample preparation on ice.

Nevertheless, our research detected all three markers selected in skeletal muscle using ELISA and succeed to provide quantitative reference values for each of them: cTnT 178.85 pg/mL, vinculin 570.96 pg/mL and desmin 565.77 pg/mL.

Limitations of PMI research in this case

We found several limitations of PMI research in the present study. Maybe the most important one is that there is only one case to investigate and report. The cause of death is another important condition that may interfere with protein degradation patterns influencing ELISA results as Bodor's study has already revealed (cTnT 0.80 mg/g protein in normal skeletal muscle, 10.0 mg/g protein in adult heart muscle, 4.37 mg/g protein in Duchenne muscular dystrophy) (38). The case reported by us has important conditional factors intricated to ictal cardiorespiratory arrest (a severe metabolic acidosis, etc) and all may interfere with protein degradation patterns which may have influenced ELISA results.

Temperature is another limitation because it may interfere with protein degradation. Temperature is important as a measure of energy which is affecting a system, accumulated degree days (ADD), (°d) = time (d) × temperature (°C) (39), and as cooling of the body until refrigeration followed by 4°C refrigeration temperature taking and a corporeal study sampling at the autopsy at room temperature in the autopsy room and temperature in the laboratory facility preparing the tissue samples (40). Body cooling time span and room temperature, refrigeration temperature, autopsy room temperature, laboratory temperature even if preparation is on ice must be scrutinized. All these limitations are to be addressed when discussing validity, reproducibility, predictability and reliability in a court of law (32, 33). Larger cohorts within this PMI range (30-40 hours PMI between hospital death and autopsy), a control group with normal, non-regenerative skeletal muscle), more extensive sampling from different muscles, may improve specificity of ELISA values. In our opinion, if we consider the specific characteristics of this case (epilepsy, GTCS, metabolic acidosis) and limitations expressed by us, these values may be used as benchmark reference values for 36.8 hour-postmortem interval.

CONCLUSIONS

The case described here highlights the important role of medico-legal autopsy to ascertain the cause of death in epilepsy. When there is an ictal death, autopsy is highly recommended. In epilepsy, autopsy death requires all documented medical data, including ECG, histopathology, etc as well toxicology examinations to clarify the manner of death (violent/non-violent).

Conditional factors may interfere in complex pathways and must be established for a correct interpretation of ictal/post-ictal cardiac involvement.

Causality is the core value of any evidence interpretation provided by a legal medicine conclusion. Limitations of any study must be taken into consideration and cautious, evidence based, objective interpretations, built on a solid scientific foundation, are recommended.

Immunoassay SDS-PAGE with Western Blot and ELISA prove to be a valuable method in PMI estimation in the skeletal muscle and detected native vinculin band 117 kD and vinculin products degradation bands 90kD, 84 kD as other cited references and additionally vinculin product band 87kD. ELISA provides quantitative values with high sensibility for cTnT 178.85 pg/mL, vinculin 570.96 pg/mL and desmin 565.77 pg/mL that may be used as reference values at 36.8-hour PMI in similar cases.

This case report supports the need for further study of PMI using immunoassay ELISA in PMI estimation: a larger casuistry with different causes of death, sampling from different muscles including regenerative / non-regenerative skeletal muscle and control group, could offer validation for ELISA values as benchmark reference within the studied PMI range.

Authors' contributions

Ioana Ruxandra Turlea – conceptualization, autopsy investigation, design, methodology, data acquisition, data validation, formal analysis, visualization, writing (original draft), final approval; George-Cristian Curca – conceptualization, design, methodology, data analysis and interpretation, formal, analysis, visualization, writing review and editing, final approval, supervision; Luminita Matei – molecular data acquisition, molecular data analysis and validation, formal analysis, visualization, review, critical revision, final approval; Alina Stoica - histopathology examination; histopathology and photo acquisition, validation, formal analysis, visualization, review, critical revision, final approval.

Institutional review board

The current study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board (Ethics Committee) of a major Institute of Legal Medicine in Romania which granted ethical approval research on postmortem interval on a series of unidentified cases including the present case (Ethics Committee Research approval registration number 3000/05.03.2024).

Informed consent

N/A (The deceased is an unidentified person. Informed consent is not required as stated by Ethics Committee Research approval registration number 3000/05.03.2024 and per national legislation Romanian Penal Procedure Code, art. 185 official medico-legal autopsy performed under a judicial order as mandatory and IRT in her capacity as a forensic pathologist appointed to perform the forensic examination).

Conflicts of interest

none declared.

Financial support

none declared.