From the *Department of Forensic Medicine and Toxicology, and †Laboratory of Clinical Biochemistry–Molecular Diagnostics, Second Department of Pediatrics, School of Medicine, University of Athens, Greece.
Manuscript received March 7, 2012; accepted December 5, 2012.
The authors report no conflicts of interest.
Reprints: Sotiris Athanaselis, PhD, Department of Forensic Medicine and Toxicology, School of Medicine, University of Athens, Mikras Asias 75, Goudi 11527, Athens, Greece. E-mail: firstname.lastname@example.org.
Venous blood cardiac troponin I (cTnI) is a sensitive biochemical marker in clinical practice for the diagnosis of myocardial infarction (MI). It is released within 6 hours after the onset of clinical symptoms. It peaks at 12 hours and remains high for at least 144 hours after the onset of symptoms.1–3 Cardiac enzymes are detectable in the pericardial fluid before they are detectable in blood in patients with MI because they reach the pericardial fluid through passive diffusion and ultrafiltration resulting from the pressure gradient.4–7 Previous studies have demonstrated the efficacy of cardiac troponin detection in postmortem biological fluids as a parameter for the postmortem diagnosis of acute MI.1,7–11
Cardiac troponin I is one of the thin filament associated regulatory muscle proteins, and it is highly specific for the diagnosis of MI. The troponin complex consists of 3 subunits and is involved in the calcium-sensitive switch that regulates the interaction of actin and myosin in striated muscles. The third subunit cTnI is the inhibitory component, and it is found exclusively in the cardiac muscle.1
The aim of the study is to evaluate the sensitivity and specificity of cTnI in pericardial fluid for the postmortem diagnosis of MI and to analyze the levels of cTnI in pericardial fluid of medicolegal autopsy cases where patients died of different causes.
MATERIALS AND METHODS
The study was approved by the ethics committee of the Medical School of the University of Athens.
Our study included 89 medicolegal autopsy cases (46 males and 43 females) selected during a 1-year period from the Department of Forensic Medicine and Toxicology of the University of Athens. The mean (SD) age of the people studied was 70.06 (17.57) years (range, 13–94 years). Cases were chosen according to the postmortem interval (<48 hours) and the cause of death. To avoid postmortem artifact, the bodies were refrigerated. The mean (SD) postmortem interval was 24.61 (10.24) hours (range, 8–48 hours). Myocardial infarction was diagnosed by histologic studies performed by a cardiac pathologist without knowledge of the respective cTnI values. In the cases studied, microscopic evidence of ischemia and/or necrosis was present.
Cases were classified into 4 groups, based on the cause of death: group A (1), myocardial infarction (n = 28); group B (2), saltwater drowning (n = 20); group C (3), death from injury in the respiratory system (injury; n = 16); and group D (4), other causes of death, excluding MI (n = 25).
Specimens were obtained from the opened pericardial sac using a sterilized syringe. Mixing with blood was avoided because hemolysis may influence the measurements.9 Hemolyzed samples were also discarded. These specimens were stored at −80°C until use. Cardiac troponin I was measured by enzyme immunoassay technique, using commercial kits from Alpco Diagnostics (Salem, NH). The principle of the assay: The cTnI ELISA test is based on the principle of a solid phase enzyme-linked immunosorbent assay. The assay system uses 4 unique monoclonal antibodies directed against distinct antigenic determinants on the molecule. The test sample is allowed to react simultaneously with the 4 antibodies, resulting in the troponin I molecules being sandwiched between the solid-phase and enzyme-linked antibodies. A solution of tetramethylbenzidine reagent is added and incubated for 20 minutes, resulting in the development of a blue color. The concentration of troponin I is directly proportional to the color intensity of the test sample. Absorbance is measured spectrophotometrically at 450 nm at the Laboratory of Clinical Biochemistry–Molecular Diagnostics, Second Department of Pediatrics, School of Medicine, University of Athens. For statistical analysis of the data, the SPSS 11.01 (SPSS Inc, Chicago, Ill) Program was used.
A probability level of P ≤ 0.05 was considered significant.
Group A included 28 cases, 18 males and 10 females (mean age, 72.11 years). The mean (SD) cTnI value was 1067.03 (889.16) mg/dL (range, 722.25–1411.81 mg/dL).
Group B included 20 cases, 7 males and 13 females (mean age, 69.35 years). The mean (SD) cTnI value was 546.98 (452.59) mg/dL (range, 335.16–758.80 mg/dL).
Group C included 16 cases, 8 males and 8 females (mean age, 73.56 years). The mean (SD) cTnI value was 398.75 (341.56) mg/dL (range, 216.75–580.76 mg/dL).
Group D included 25 cases, 13 males and 12 females (mean age, 66.08 years). The mean (SD) cTnI value was 577.47 (634.16) mg/dL (range, 315.70–839.23 mg/dL).
Using nonparametric test (Table 1), we compared the mean values of cTnI among the 4 diagnostic groups A, B, C, and D.
Group A had a significant difference regarding the mean concentration of cTnI when compared with the other groups.
In group A, we obtained statistically significant differences in relation to groups B, C, and D.
Table 2 presents the P values of the comparisons among the aforementioned groups and the differences of their averages. Because all P values are less than 5%, it becomes clear that the mean concentration of cTnI for the cases of group A differs significantly from those of the other groups B, C, and D in the 5% significance level.
There were no statistically significant differences between sexes, regardless of the cause of death (Fig. 1).
Furthermore, we observed that the cTnI values peak between the ages of 55 and 74 years. For ages older than 75 years, cTnI mean value seems to be decreased but does not approximate the levels of cTnI in the ages younger than 55 years (Fig. 2).
Myocardial infarction is a major cause of death and disability worldwide. In pathology, it is defined as myocardial cell death due to prolonged ischemia.
After the onset of myocardial ischemia, cell death is not immediate but takes a finite period to develop. Thus, it takes several hours before myocardial necrosis can be identified by macroscopic or microscopic postmortem examination.12 Myocardial infarction is diagnosed when blood levels of sensitive and specific markers such as cardiac troponin or creatine kinase–MB are increased in the clinical setting of acute myocardial ischaemia.13 The preferred biomarker for the diagnosis of myocardial necrosis is cardiac troponin (I or T). It has nearly absolute myocardial tissue specificity as well as clinical sensitivity, thereby reflecting even microscopic zones of myocardial necrosis.14
On several occasions in forensic practice, it is difficult to diagnose acute myocardial infarct based only on pathologic observations. In such cases, complementary diagnostic techniques, such as the determination of biochemical markers in postmortem biological fluids, could be of outmost importance.9 Research is directed toward a biochemical marker, specific enough for the postmortem diagnosis of myocardial cellular lesion. The aim of several researched markers is to provide high concentrations in myocardial injury and trace amounts in other pathologic conditions.15,16 This last characteristic would be of interest in clinical practice but not in postmortem diagnosis, where any marker should also be free of interference arising from the period during the postmortem interval and from contamination caused by adjacent fluids.1 An ideal marker for diagnosis of acute MI should be cardiac specific to allow reliable diagnosis of myocardial damage in the presence of skeletal muscle injury; it should be highly sensitive and should detect even small damage.9
In this study, the pericardial fluid was studied because of its unique characteristics, among which, it is the close proximity to myocardial tissue, which means that any alterations in the cardiac muscle would be detected at an earlier stage in this fluid rather than in the serum.9 We obtained statistically significant differences in pericardial fluid for cTnI, the highest levels being obtained in the group where the cause of death was MI. These results correlate with similar previous studies.1,9 For discriminant analysis, we used the cause of death as the grouping variable, establishing 2 groups: deaths caused by MI (28 cases) and cases with no MI (61 cases). The mean concentration of cTnI of the first group differs significantly from the second group in the 5% significance level. We observed that the cTnI values peak between the ages of 55 and 74 years. For ages older than 74 years, cTnI value seems to be decreased but does not approximate the levels of cTnI observed in the ages younger than 55 years (Fig. 2). No statistically significant correlations were observed between the levels of cTnI and the postmortem interval. A cutoff level of cTnI mean concentration could not be determined because there is an overlap between group A and groups B and D.
It becomes clear that cTnI is a very useful marker of myocardial damage and should complement histologic studies to establish an accurate diagnosis of MI in postmortem samples, although postmortem interference should be taken under consideration. Thus, research including a bigger number of cases and a larger variability of causes of death could be useful to determine a cutoff level of cTnI.
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