Sudden cardiac death, often attributed to acute myocardial infarction (AMI), remains a significant concern in forensic pathology. Despite advances in diagnostic modalities, the postmortem identification of early myocardial infarction—especially within the first few hours—continues to pose a diagnostic challenge due to the absence of definitive histological changes in the myocardium during the early phase (1). Traditional histopathological techniques may not reliably detect early ischemic alterations, necessitating the need for alternative diagnostic approaches.

Biochemical markers have emerged as a vital adjunct in the postmortem diagnosis of AMI. Cardiac troponins, particularly troponin I (cTnI), are highly specific to myocardial tissue and are considered the gold standard in clinical settings for the diagnosis of myocardial injury (2). Other markers such as creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) also provide useful information when interpreted in conjunction with clinical or pathological findings (3). However, the postmortem analysis of these biomarkers in peripheral blood is often complicated by hemolysis, redistribution phenomena, and autolytic changes (4).

Given these limitations, the analysis of alternative body fluids, such as vitreous humor and pericardial fluid, is gaining interest. Vitreous humor is relatively protected from postmortem changes due to its anatomical isolation and low enzymatic activity, making it a stable medium for biochemical analysis even several hours after death (5). Pericardial fluid, due to its anatomical proximity to the heart, may offer an even earlier and more accurate reflection of myocardial damage compared to peripheral blood (6). Studies suggest that these fluids may provide complementary information and improve the sensitivity and specificity of postmortem biochemical diagnosis of AMI (7,8).

In this context, the present study aims to evaluate and compare the efficacy of vitreous humor and pericardial fluid in detecting early myocardial infarction postmortem through the analysis of cardiac biomarkers including cTnI, CK-MB, and LDH. The goal is to enhance diagnostic accuracy and support forensic investigations in cases of sudden, unexplained death where myocardial infarction is suspected.

A total of 60 autopsy cases were included based on specific inclusion and exclusion criteria. The study population was divided into two groups:

Group A (Myocardial Infarction group): 30 cases with confirmed acute myocardial infarction based on gross pathological findings and/or clinical history.

Group B (Control group): 30 cases with non-cardiac causes of death such as trauma, hanging, or poisoning, with no evidence of cardiac pathology.

Only cases with a known postmortem interval (PMI) of less than 12 hours were considered, to minimize the impact of autolysis on biomarker stability. Cases with advanced decomposition or those where sample collection was not feasible were excluded.

Sample Collection:

During autopsy, approximately 2–3 mL of pericardial fluid was aspirated using a sterile syringe from the pericardial cavity before organ dissection. Vitreous humor was obtained by aspirating 1–2 mL from the posterior chamber of the eye using a 22-gauge needle under aseptic conditions. All samples were collected within the first 12 hours of death and transferred to sterile containers.

Biochemical Analysis:

Samples were centrifuged at 3000 rpm for 10 minutes and the supernatant was stored at -20°C until analysis. Quantitative determination of cardiac troponin I (cTnI), creatine kinase-MB (CK-MB), and lactate dehydrogenase (LDH) levels was performed using a chemiluminescent immunoassay method on a fully automated analyzer. All assays were conducted in accordance with the manufacturer’s instructions and under standard laboratory conditions.

Statistical Analysis:

Data were entered into Microsoft Excel and analyzed using SPSS version 25.0 (IBM Corp, Armonk, NY). Descriptive statistics such as mean and standard deviation were calculated. Comparative analysis between groups was performed using the independent t-test for normally distributed data. Receiver Operating Characteristic (ROC) curves were plotted to evaluate diagnostic accuracy. A p-value of less than 0.05 was considered statistically significant.

The present study analyzed biochemical markers—cardiac troponin I (cTnI), creatine kinase-MB (CK-MB), and lactate dehydrogenase (LDH)—in pericardial fluid and vitreous humor among 60 postmortem cases. The distribution of marker levels was compared between the myocardial infarction (Group A) and control (Group B) groups.

As shown in Table 1, the mean cTnI level in pericardial fluid for Group A was significantly elevated (6.82 ± 1.87 ng/mL) compared to Group B (1.21 ± 0.44 ng/mL), with a p-value < 0.001. Similarly, vitreous humor cTnI levels were higher in Group A (3.46 ± 1.15 ng/mL) than in Group B (0.89 ± 0.31 ng/mL), also reaching statistical significance (p < 0.001).

Table 1: Comparison of cTnI levels in Pericardial Fluid and Vitreous Humor between Study Groups

CK-MB levels followed a similar pattern, as shown in Table 2. Group A had higher mean CK-MB values in pericardial fluid (48.5 ± 9.3 U/L) compared to Group B (15.7 ± 4.8 U/L; p < 0.001). In vitreous humor, the values were 29.4 ± 7.8 U/L and 9.6 ± 3.1 U/L for Groups A and B respectively (p < 0.001).

Table 2: Comparison of CK-MB Levels between Study Groups

For LDH, as presented in Table 3, Group A showed significantly higher enzyme activity in pericardial fluid (1024.6 ± 134.2 U/L) than in Group B (410.3 ± 95.6 U/L; p < 0.001). Vitreous LDH levels were also significantly different (Group A: 612.8 ± 110.4 U/L; Group B: 278.7 ± 84.9 U/L; p < 0.001).

Table 3: Comparison of LDH Levels in Study Groups

Receiver Operating Characteristic (ROC) analysis indicated that cTnI in pericardial fluid had the highest diagnostic accuracy (AUC = 0.93), followed by cTnI in vitreous humor (AUC = 0.87), demonstrating their superior sensitivity and specificity in detecting early myocardial infarction postmortem

The early postmortem diagnosis of acute myocardial infarction (AMI) remains a considerable challenge in forensic medicine, particularly when gross morphological and histopathological features are inconclusive. The present study demonstrates that biochemical markers, especially cardiac troponin I (cTnI), measured in pericardial fluid and vitreous humor, can serve as reliable indicators of early myocardial injury.

Our findings revealed significantly elevated levels of cTnI, CK-MB, and LDH in both pericardial fluid and vitreous humor among individuals who died of confirmed myocardial infarction, compared to non-cardiac controls. Among these, cTnI in pericardial fluid showed the highest diagnostic accuracy. This observation aligns with earlier studies which have established cardiac troponins as highly specific and sensitive indicators of myocardial cell damage, even in postmortem scenarios (1,2).

Pericardial fluid, due to its close anatomical relation to the myocardium, is believed to accumulate biomarkers rapidly after cardiac injury. Studies by Palmiere et al. and Sajjad et al. have highlighted its utility in postmortem diagnostics, reporting high troponin concentrations in pericardial fluid in MI cases compared to peripheral blood or other fluids (3,4). Our findings corroborate these results, reinforcing the diagnostic potential of pericardial fluid analysis in forensic settings.

Vitreous humor, though anatomically distant from the heart, offers a distinct advantage due to its resistance to postmortem degradation and microbial invasion (5). Several researchers have advocated its use in the biochemical investigation of postmortem cases, citing its stability for a variety of markers, including urea, glucose, and electrolytes (6,7). In our study, although vitreous humor yielded slightly lower concentrations of cardiac markers compared to pericardial fluid, the differences between groups remained statistically significant, confirming its value in cases where pericardial access is not feasible.

The diagnostic performance of LDH and CK-MB was also noteworthy, though less specific than cTnI. CK-MB, while considered more specific than total CK, may be influenced by skeletal muscle injury or prolonged postmortem intervals (8). LDH, being a ubiquitous enzyme, can also increase in various systemic conditions or due to tissue autolysis (9). Nevertheless, when interpreted in conjunction with cTnI levels, these enzymes provide supportive diagnostic evidence.

Time since death (postmortem interval) is an important variable that affects the stability of biochemical markers. Previous studies have shown that cTnI levels remain relatively stable for several hours postmortem, particularly in controlled storage conditions (10,11). In our study, only cases with a PMI under 12 hours were included to minimize autolytic changes and preserve biomarker integrity, which likely contributed to the clarity of results.

Despite the promising findings, this study has some limitations. The sample size was modest, and biochemical analyses were confined to a single time-point postmortem. Larger-scale studies with serial sampling and consideration of different environmental and physiological conditions could provide further insights. Additionally, integrating immunohistochemical detection of cardiac markers in tissue sections may enhance diagnostic accuracy (12,13).

Overall, our findings emphasize the importance of using alternative body fluids for postmortem biochemical analysis. The combination of cTnI measurements in pericardial fluid and vitreous humor offers a valuable adjunct in the forensic assessment of sudden cardiac deaths, especially when conventional autopsy findings are non-contributory.

Future research should focus on standardizing cut-off values for postmortem cardiac markers in various fluids and developing comprehensive forensic protocols that integrate biochemical, histological, and molecular tools (14,15).

The analysis of postmortem biochemical markers in pericardial fluid and vitreous humor, particularly cardiac troponin I, provides valuable diagnostic insight into early myocardial infarction. Pericardial fluid offers the highest diagnostic accuracy due to its proximity to the heart, while vitreous humor serves as a reliable alternative in decomposed or challenging cases. Integrating these biochemical evaluations with immunohistochemical findings can significantly enhance the accuracy of postmortem diagnosis in sudden cardiac deaths.

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