POSTMORTEM ASSESSMENT OF CARDIAC TROPONIN-T (TROPOMYCIN) AND CARDIAC TROPONIN-I (INHIBITOR) LEVELS TO DIAGNOSE MYOCARDIAL INFARCTION IN VICTIMS OF SUDDEN CARDIAC DEATHS

Background: Sudden cardiac death (SCD), which is caused by acute myocardial infarction (AMI), represents a considerable percentage of autopsy cases. In many situations in forensic medicine, the diagnosis of acute myocardial infarction based only on pathological findings is difficult, so the determination of biochemical markers in postmortem biological fluids may be of significant value. Objective: The present study has aimed to throw light upon the role of postmortem assessment of cardiac troponin-T and cardiac troponin-I levels in the diagnosis of AMI in SCDs. Methodology: This is a prospective study that was conducted on 50 deceased (who fulfilled inclusion criteria) of sudden death and a control group of 20 cases consisting of deaths due to causes other than sudden death, including poisoning, falling from height, and stab wounds, that were admitted to the Forensic Medicine Authority, Alexandria Department, from January 2018 to March 2019 for comparison. At the time of autopsy, hearts were exposed, from which blood was obtained during internal examination of the thoracic cavity. About 10 ml of blood was obtained from the heart left chamber by a sterile syringe and was used for the analysis of cardiac troponin T and cardiac troponin I levels after centrifugation to obtain plasma. Results : All the assessed parameters showed significant correlation with SCDs. The sensitivity of cTnI and cTnT for prediction of SCD was 100% with a specificity of 85%. Conclusion: Evaluation of the biocardiac enzymes of cTnI and cTnT represents a highly sensitive marker in postmortem determnination of myocardial lesions in SCD.


INTRODUCTION
The incidence of sudden cardiac death (SCD) has been steadily rising worldwide, now accounting for annual deaths of over 17 million persons, which constitutes about a third of mortality globally.Identifying the cause of SCD is a priority for physicians handling these cases; however, it presents a significant challenge for forensic pathologists.Acute myocardial infarction (AMI) is globally recognized as one of the principal causes of death and a major threat to human life.In clinical settings, the diagnosis of AMI is facilitated by electrocardiograms and specific serum biochemical markers that detect myocardial damage (Han and Flavin, 2014; Taniguchi et al., 2016).
In forensic pathology, SCD is recognized as a frequent cause of acute mortality.While conventional pathological techniques can readily identify typical myocardial lesions, quantitatively assessing the severity of myocardial damage remains challenging.In cases of sudden death occurring at the very early stages of infarction, positive pathological evidence is often absent, necessitating a diagnosis based on the exclusion of other potential causes of death (Yasuda et al.,

2005).
The autopsy is well known to be limited in diagnosing mortality caused by ischaemic heart disease (IHD) and is well recognized.Much research has explored using biochemical analysis to detect levels Egypt J. Forensic Sci.Appli.Toxicol.
Vol 24 (3), September 2024 of cardiac troponins in postmortem bodies as a method to differentiate cardiac from non-cardiac mortality (Davies et al.,

2005).
In medico-legal casework, assessing myocardial damage is a crucial aspect of postmortem investigations for various causes of death.Recent trends in applying biochemical methods to forensic pathology have shown significant interest in utilizing cardiac troponins (proteins specific to cardiac myofibrils) for postmortem examinations of IHD, which is mainly implicated in sudden death in several populations (Zhu et al., 2006).
Troponin is a complex protein that is located on the actin filament, regulating the actin-myosin interaction during muscle contraction.Troponin is constituted of three subunits: tropomyosin binding (cTnT), inhibitory (cTnI), and calcium binding (TnC).It is typically absent from the serum unless there has been necrosis of cardiac cells, making it highly specific to cardiac tissue.Current research has demonstrated that the levels of cardiac troponins act as a reliable marker of AMI (Bheeshma et al., 2015).
The current study aimed to evaluate the use of postmortem cardiac troponin T (cTnT) and cardiac troponin I (cTnI) levels for diagnoses of acute myocardial infarction in medicolegal autopsy and correlate them with the histopathological findings.

SUBJECTS & METHODS A) Subjects:
This is a prospective study that was conducted on 50 deceased (who fulfilled inclusion criteria) of sudden death and a control group of 20 cases consisting of deaths due to causes other than sudden death, including poisoning, falling from height, and stab wounds, that were admitted to the Forensic Medicine Authority, Alexandria Department, from January 2018 to March 2019 after an official permission from the director of the Alexandria Medical Authority, scientific and ethical committee with ethical approval number; Ms. 16.1.2018.

Inclusion Criteria:
 Sudden death of an adult with the cause of death unexplained by external examination (body found deceased at home, outdoors, or in the hospital emergency room).

B) Methods:
Autopsy was performed with a single midline incision that extended from the chin to the pubic bone, and then the skin, muscles, and soft tissue were peeled back, exposing the rib cage, which was pulled from the skeleton.
The abdomen was further opened by dissecting the abdominal muscles away from the bottom of the rib cage and diaphragm.The organs were removed individually, but before that, each organ was examined in situ for any gross pathology.For removal of the opening in, an incision was made from one ear to the other: through the vertex, the scalp was cut and then pulled forward and backward, and the open of the skull was removed by using a vibrating saw.Then the brain was exposed and examined in situ before removal.After removal of the body organs, each one was weighted.At the time of autopsy, hearts were exposed, from which blood was obtained during internal examination of the thoracic cavity.Approximately ten mililiters of blood were obtained from the heart's left chamber using a 21G sterile syringe and needle.The blood was then placed in a test tube and centrifuged immediately to obtain plasma at a frequency of 3,000 rpm.The obtained plasma was stored at 4°C for no more than 24 hours before being sent to Egypt J. Forensic Sci.Appli.Toxicol.
Vol 24 (3), September 2024 the department laboratory for analysis to measure levels of cardiac troponin T and cardiac troponin I.

A-Biochemical analysis:
Cardiac troponin T was measured using Elecsys Troponin T kits, and cardiac troponin I was measured using Elecsys Troponin I kits; both were measured using Cobas e 601 analyzers by the Sandwich principle, according to Anderson (2013).

B-Histopathological study:
After removal of the heart, a gross examination was done for any pathology, and then it was put in a container filled with 10% formalin.The containers were covered, labeled, and sent for histopathological examination.For histopathological studies, formalin-fixed paraffin 5 micrometer sections from the heart were stained with hematoxylin and eosin (H & E) according to Lamberg & Rothstein (1978) for examination by light microscope.

Statistical Analysis:
The collected data were organized, tabulated, and statistically analyzed using IBM SPSS statistical software, version 20.0 (Armonk, NY: IBM Corp).Categorial variables were expressed in numbers and percentages (Kirkpatrick and Feeney, 2012), and the data normality was tested using Kolmogorov-Smirnov.Numerical data were presented in median, range (minimum-mmaximum), mean, and standard deviation.Student t-test, chi square (2), ANOVA (F-test), Fisher exact test, Monte Carlo (MC) correction, Kruskal-Wallis test, Spearman coefficient, Pearson correlation coefficients, and post hoc test.The accepted level of significance of the obtained results was stated at 0.05.Sensitivity and specificity were assessed.

a-Sensitivity
Sensitivity assesses the percentage of the correctly determined actual positive cases (Tom, 2006).In this study, we calculate sensitivity according to the following formula:

b-Specificity
Specificity assesses the percentage of correctly determined actual negative cases (Tom, 2006).In our study, we calculated sensitivity according to the following formula:

RESULTS
This study included 70 cases who were divided into two groups as regard the cause of death: group (I) included 50 (71.4%)cases who had sudden cardiac death, and group (II) the control group included 20 (28.6) cases who died due to other causes such as poisoning [7 (10%) cases], falling from height [8 (11.42%) cases], and stab wound [5 (7.14%) cases] as shown in Table 1 and fig. 1.The results of the current study illustrated a higher prevalence in the number and percentage of males among both the studied and control cases.However, the MI studied group experienced a statistically insignificant difference in the gender distribution as compared with the control group (Table 2  and fig.

2).
Data analysis among the MI studied group showed a higher prevalence of sudden cardiac deaths in the seventh-and eighth-decade age group (age between 60-70 and 70-80 years old), as shown in Table 3.The mean age of MI and the control group was 61.14 ± 12.79 (range 34.0 -81.0years) and 33.15± 13.12 (range 16.0 -57.0 years), respectively.Statistical analysis of the previous data showed significant differences in the mean age between the control group and the MI studied group (Table 4).
Vol 24 (3), September 2024 In our study, the biochemical analysis results of cTnI and cTnT documented a markedly significant increased level of cTnT and cTnI in MI cases compared to control.The mean values of cTnT and cTnI were [752.0 ±271.6 (range: 190.0 -1310) and 1711.2 ± 1024.4 (range: 213.0 -3519.0),respectively, among the MI group (Table 5).
The histopathological findings revealed that infarction was documented in the whole MI cases 50, where 43 of them (86%) had acute MI evidenced by wavy myocardiocytes that signify necrosis or cell death and inflammation caused by necrosis, which is characterized by the existence of several immune cells, particularly neutrophils, and 7 (14%) cases had old MI evidenced by replacing the necrotic myocardium with fibrous and vascular tissue.Whereas in the control group, old MI was evident only in 3 (15%) cases, with a significant difference between the two groups regarding prevalence or absence of MI (Table 6 and fig.3, 4 & 5).
The findings of the current study showed that the sensitivity of both cTnT and cTnI for prediction of sudden cardiac death was 100% with a specificity of 85% (Table 7).
Considering studying the relation between cTnT levels and cardiac histopathology (Table 8 and fig.6) and between cTnI levels and cardiac histopathology (Table 9 and fig.7) in both the MI group and the control group, there was a significant difference in the mean values of cTnT regarding histological findings between both groups.Additionally, the results revealed a significant positive moderate correlation between both cTnT and cTnI in the MI group (rs = 0.591, p < 0.001), meaning the correlation was insignificant in the control group, as shown in Table 10.
Declaring the postmortem interval (PMI), the present study results illustrated that PMI in the MI group ranged from 7 to 43 hours with a mean SD of 24.12 to 10.51, while the control group ranged from 8 to 41 hours with a mean SD of 24.05 to 9.09, with an insignificant difference between both groups as demonstrated in Table 11.
As regard correlation between PMI and both cTnT and cTnI levels, there was insignificant weak to fair positive correlation between PMI and cTnT levels in both MI groups and in the control group (r = 0.058 and r = 0.225, respectively).However, this correlation with cTnI was insignificantly weak positive with time interval in the MI group and control group (r = 0.094 and r = 0.231, respectively, as demonstrated in Table 12.
Vol 24 (3), September 2024       Cardiac troponin I (cTnI), being exclusively found in the myocardium, serves as a highly specific biomarker of myocardial injury.Postmortem serum and pericardial fluid levels of cTnI are significantly elevated in individuals who experienced acute myocardial ischaemia compared to control subjects (Belsey & Flanagan, 2016).
Data analysis of the current study among the MI group showed a higher prevalence of sudden cardiac death in the seventh-and eight-decade age group between 60-70 and 70-80 years and a higher prevalence in the number and percentage of male cases as compared to females with the MI studied group, which experienced a statistically insignificant difference in the gender distribution as compared with the control group.The results of this study revealed that infarction was evident by histopathology in all cases in the MI group, whereas in the control group, old MI was evident in 3 (15%) cases, with a significant difference between the two groups.
Regarding biochemical analysis, these results showed that cTnI and cTnT had significantly higher values in MI cases than control cases.These findings are in accordance with Carvajal-Zarrabal et al. ( 2017), who measured cTn-I and cTn-T in blood samples from the pulmonary veins of twenty dead bodies; those were clinically diagnosed to have SCD, and eight bodies with variable causes of violent death found that cTn-I and cTn-T levels were significantly elevated in cardiac versus noncardiac deaths.Further, Peter et al.'s (2006) study measured troponins cTnT and cTnI in twenty-five cases diagnosed with cardiac injury and eleven control cases without significant trauma to myocardium and revealed that there are significant differences in the cases with cardiac injury and those without for cardiac troponin T & I.In contrast, Rahimi et al. ( 2018) reported no significant difference in cTnT level measured in 140 cases with PMI less than 24 hours and had different causes of death (p ≥ 0.05) such as CVD, thoracic or nonthoracic injury, sudden unexplained death, and others.This may have been attributed to microangiopathies and increased permeability of the myocardial cells; oxygen supply is reduced, and demand is increased.Also, Bheeshma et al. (2015) studied 12 cases with myocardial infarction and 6 control cases and showed that troponin T was elevated (>2.000 ng/ml) in all cases of sudden death except one case and in control cases except 2 cases; however, the p value was not studied for comparison, which disagrees with these results.The correlation between both troponins was significant, positive, and moderate in the MI group while the correlation was insignificant in the control group.This significant correlation may be due to the same kinetics of cTnT and cTnI, as they are released within 6 hours of a myocardial cellular lesion and persist in plasma for several hours.No studies have been done to investigate the correlation between both troponins for comparison.
Concerning the relation between cTnT and cTnI levels and pathology, the results showed that there was a significant difference between both in the MI group and the control group; this goes in line with Bheeshma et al. ( 2015 2006) also reported that all bodies with morphologically apparent myocardial infarction had increased cTnT levels, which agree with these results.
Regarding PMI, in the MI group it ranged from 7 to 43 hours, while the control group ranged from 8 to 41 hours, with a significant difference between both groups.Studying the correlation of cTnI and cTnT with PMI demonstrated insignificant weak to fair positive correlation in both groups.Increased troponin T and I levels with time interval occur as autolysis causes cellular membrane damage, which leads to leakage of troponin outside of cells, but this increase is insignificant (p > 0.05) as revealed by these results.2016), who studied cTnT levels in pericardial fluid and serum obtained from fifty-eight cases at defined postmortem intervals and concluded that cTnT showed stability in levels up to 34 hours after death.2013), who studied postmortem cTnT in the serum and pericardial fluid of 101 autopsied cases with PMI of 8-141 h, reported a significant positive correlation between pericardial fluid and serum cTnT level and PMI, which disagrees with these results; this may be attributed to the long PMI of cases in their study.

CONCLUSIONS
In view of the previous results, it could be concluded that:  To properly evaluate the cardiac lesions of persons who died suddenly, including cases where death involves myocardial infarction, you have to estimate both cTnI and cTnT.Using this procedure, it should be possible to rule out a cardiac cause of death with high accuracy, i.e., evaluation of the biocardiac enzymes of cTnI and cTnT represents a highly sensitive marker of myocardial lesion.
 Determination of cTnI and cTnT levels will be important to exclude false positive results of diagnosing cases of Figure (2): Comparison between the studied cases according to sex Figure (3):Comparison between the two studied groups according to histopathological findings.

Figure ( 5 ):
Figure (5): Old MI shows replacement of necrotic cardiac muscle tissue with vascular and fibrous tissue.
These results agree with Peter et al. (2006), who concluded that cardiac blood cTnI exhibited a nonsignificant positive correlation with the time of autolysis; also, Vanhaebost et al. (2017) proved that there were no statistically significant correlations between troponin T & I concentrations collected from peripheral blood (femoral vein) and postmortem interval.Contrary, these results disagree with Gonza´lez-Herrera1 et al. (

Table ( 1
): Distribution of the studied cases according to cause of death.
Figure (1): Distribution of the studied cases according to cause of deathTable (2): Comparison between the studied cases according to sex.

Table ( 3
): Comparison between MI studied and control cases according to different age groups.Chi square test, MC: Monte Carlo, p: p value for comparing between the studied groups, *: Statistically significant at p ≤ 0.05.

Table ( 5
): Comparison between the two studied groups according to post mortem troponin level.

Table ( 6):
Comparison of the two studied groups according to histopathological findings.

Table ( 8
): Relation between cTnT and pathology among both MI & control group.

p: p value for comparing between the studied groups Table (12): Correlation
between postmortem interval (PMI) and both cTnT and cTnI among the studied groups.