Forensic Science International 233 (2013) 154–157
Contents lists available at ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Cardiac troponin T in forensic autopsy cases S. Remmer a,b,*, A. Kuudeberg a, M. To˜nisson a, D. Lepik a, M. Va¨li a,b a b
Institute of Pathological Anatomy and Forensic Medicine, University of Tartu, Ravila 19, 50411 Tartu, Estonia Estonian Forensic Science Institute, Tervise 30, 13419 Tallinn, Estonia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 May 2013 Received in revised form 4 September 2013 Accepted 6 September 2013 Available online 14 September 2013
The aim of the present study was to describe the findings of postmortem serum and pericardial fluid (PF) cardiac troponin T (cTnT) in various causes of death with regard to the postmortem interval (PMI) and comorbid cardiovascular disease, using 101 autopsy cases with PMI of 8–141 h divided into 9 groups: cardiovascular disease (CVD), other diseases (OD), poisoning (P), asphyxia (A), drowning (D), hypothermia (H), thoracic trauma (TT), other trauma (OT) and fire fatalities (F). The results suggest that cTnT levels may help to differentiate cardiovascular death from poisoning and non-thoracic trauma, as well as to differentiate cardiovascular and other diseases as cause of death from drowning and hypothermia. However, the effect of PMI, unlike comorbid cardiovascular disease, has to be taken into account. ß 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Postmortem biochemistry Cardiac troponin T Blood Pericardial fluid
1. Introduction The use of cardiac troponins as a part of laboratory investigations in forensic autopsy has been analysed by several authors [1– 11]. Cardiac troponin T (cTnT), a marker for myocardial injury, has been recommended for the evaluation of the presence and extent of myocardial damage in various causes of death [12–15]. cTnT may be used to confirm the diagnosis of cardiac-related death and in distinguishing certain causes of deaths from others [12,13,15– 17]. In the living, an increase in the level of cTnT in serum can be seen approximately 2–4 h after the onset of cardiac symptoms and persists for up to 2 weeks [18]. Postmortem cTnT levels depend on several factors, such as the severity and duration of myocardial damage before death, the cause of death, the postmortem interval (the time between death and the collecting of samples, PMI) and the sampling site [6,8,9,12–14,19–21]; therefore, clinical serum cTnT reference values cannot be directly used in interpreting postmortem samples and cut-off values for postmortem cTnT levels have been suggested [12,13,16]. Besides acute myocardial infarction, other conditions, especially cardiovascular diseases, are known to result in elevated cTnT levels [3,12,13,16,18], and the effect of comorbid disease on postmortem cTnT levels should be
* Corresponding author at: Institute of Pathological Anatomy and Forensic Medicine, University of Tartu, Ravila 19, 50411 Tartu, Estonia. Tel.: +372 7374290. E-mail addresses: [email protected] (S. Remmer), [email protected] (A. Kuudeberg), [email protected] (M. To˜nisson), [email protected] (D. Lepik), [email protected] (M. Va¨li). 0379-0738/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.forsciint.2013.09.010
taken into account. Due to rapid postmortem putrefaction of blood, the use of alternative biological fluids, such as pericardial fluid (PF) is advised [8,19,20,22,23]. There is no previous experience using postmortem cardiac markers as a diagnostic tool in forensic autopsies in Estonia. The aim of this study is to describe the findings regarding the possible use of postmortem serum and pericardial fluid cTnT in investigating the cause of death. 2. Materials and methods 2.1. Materials A total of 230 forensic autopsy cases at the Estonian Forensic Science Institute were examined. At autopsy, blood was drawn from femoral/iliac veins and pericardial fluid was collected after opening the pericardial sac. PF samples contaminated with blood and all cases with evident decomposition were excluded. The final sample consisted of 101 cases (87 males and 14 females), 25– 54 years of age with a median of 42 years of age. The estimated postmortem interval was 8–141 h with a median of 34.5 h. The cause of death was grouped on the basis of gross, histological and toxicologial findings as follows: cardiovascular disease (CVD, n = 10), other diseases (OD, n = 12), poisoning (P, n = 20), asphyxia (A, n = 25), drowning (D, n = 11), hypothermia (H, n = 5), blunt and sharp thoracic trauma (TT, n = 7), other trauma (OT, n = 8) and fire fatalities (carbon monoxide poisoning, F, n = 3). The CVD group included cases with International Classification of Diseases codes I20–25 and I 30-52. The poisoning group consisted of cases with acute lethal poisoning by the following agents: ethanol, surrogate alcohol, diethyl ether, diazepam, phenobarbital, pentobarbital, thiopental, clozapine, tramadol, fentanyl, 3-methylfentanyl, methadone and amphetamine. All cases were classified according to the presence or absence of comorbid chronic cardiovascular disease (n = 36 and n = 65, respectively). PMI was divided into 5 groups: less than 12 h (n = 8), 12–24 h (n = 20), 24–48 h (n = 41), 48–72 h (n = 21) and over 72 h (n = 11). Case profiles are shown in Table 1. Due to the small number of cases, fire fatalities were left out of further statistical analysis.
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Table 1 Case profiles (n = 101, 87 male and 14 female). Cause of death
n
Age (years)
PMI (h)
Range
Median
Range
Median
CVD OD P A D H TT OT F
10 12 20 25 11 5 7 8 3
28–53 30–54 27–52 41–54 25–51 27–54 26–54 29–54 43–51
48.0 42.5 40.0 40.0 39.0 48.0 37.0 42.5 48.0
16.5–100.0 11.0–129.5 9.0–84.0 8.0–96.0 13.5–57.5 22.0–106.0 8.5–66.3 8.0–74.0 10.5–141.0
34.3 34.0 35.0 36.0 35.5 35.0 16.0 25.8 106.0
CVD, cardiovascular disease; OD, other diseases; P, poisoning; A, asphyxia; D, drowning; H, hypothermia; TT, thoracic trauma; OT, other trauma; F, fire fatality; PMI, postmortem interval (h) hours.
2.2. Biochemical analyses Samples were stored at +4 8C during transportation to the laboratory (24 h). Blood was centrifuged to separate the serum. Samples were stored at 20 8C until use. cTnT was measured using electro-chemiluminescence immunoassay (Roche Diagnostics Elecsys). The upper limit for clinical reference range was 0.03 ng/ml. 2.3. Statistical methods Spearman’s rank order correlation (rs) was used to evaluate the relationship between pairs of parameters. Non-parametric Kruskal–Wallis test was used to compare groups and the non-parametric Mann–Whitney U test was used for comparisons between individual groups. Statistical analysis was performed using statistical software R (version 2.14.0) and Statistica 12.0. A p-value of less than 0.05 was considered statistically significant.
3. Results We found no significant relationship between cTnT levels and age or gender. There was a positive correlation between serum cTnT and pericardial fluid cTnT levels (rs = 0.43, p < 0.001, Spearman). The level of pericardial fluid cTnT was higher compared to serum cTnT among all groups of causes of death, except for asphyxiation and hypothermia. When compared pairwise, pericardial fluid cTnT levels were higher compared to serum cTnT levels in most cases (90/101) and equal in 1 case. In cases with higher serum cTnT compared to pericardial fluid cTnT, the cause of death was asphyxiation in 5 out of 10. We found a moderate positive correlation between pericardial fluid cTnT level and PMI (rs = 0.41, p < 0.001, Spearman) and a weak positive correlation between serum cTnT and PMI (rs = 0.22, p = 0.02, Spearman). A significant postmortem time-dependent elevation in serum and pericardial fluid cTnT levels emerged between different PMI groups (p-value = 0.04 for serum and pvalue = 0.004 for pericadial fluid, Kruskal–Wallis, Figs. 1 and 2). Among different groups of COD, postmortem time-dependent elevation in pericardial fluid cTnT levels was found for asphyxiation (rs = 0.80, p-value < 0.001, Spearman). No statistically important postmortem elevation in serum cTnT levels for COD groups was noticed.cTnT levels in serum and pericardial fluid regarding different causes of death are shown in Table 2. Serum cTnT levels in the CVD group were significantly higher compared to poisoning (pvalue < 0.01, Mann–Whitney U test). Pericardial fluid cTnT levels showed significantly higher results in the CVD group compared to drowning (p-value < 0.01), other trauma (p-value = 0.03) and hypothermia (p-value < 0.01). Also, pericardial fluid cTnT levels were significantly higher in OD group compared to drowning (pvalue = 0.02) and hypothermia (p-value = 0.03). We found no significant difference in the levels of serum and PF cTnT between cases with and without CVD comorbidity for all COD
Fig. 1. Serum cTnT levels according to PMI interval.
(p-value = 0.7549 for serum, p-value = 0.3402 for pericardial fluid, Kruskal–Wallis) or among individual COD groups (p-value = 0.7575 and p-value = 0.342 respectively, Mann–Whitney Utest, Table 3).
4. Discussion Cardiac troponin T (cTnT) has previously been assessed for the postmortem diagnosis of myocardial injury with some controversy. Postmortem cTnT levels are generally lower in serum from peripheral blood compared to the levels in pericardial fluid, and the proximity of the myocardium and direct leakage of cTnT into the pericardium has been suggested as the cause [12,13,24]. Correspondingly, pericardial fluid cTnT levels were generally higher compared to serum cTnT levels in our study. We found a positive correlation between the cTnT levels in serum and pericardial fluid, which has not been previously reported. A postmortem time-dependent elevation in cTnT levels in the first days after death has been shown previously [6,9,12,13],
Fig. 2. Pericardial fluid cTnT levels according to PMI interval.
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Table 2 Serum and pericardial fluid cTnT regarding cause of death. Cause of death
n
CVD OD P A D H TT OT F
10 12 20 25 11 5 7 8 3
Serum cTnT (ng/ml)
PF cTnT (ng/ml)
Range
Median
Range
Median
0.03–84.36 0.01–214.70 0.01–80.96 0.01–500.00 0.01–17.54 0.01–24.02 0.01–39.28 0.01–13.66 0.01–0.81
10.56 1.10 0.21 0.60 0.83 0.28 0.20 0.55 0.06
4.98–250.00 0.51–310.40 0.04–192.70 0.19–271.90 0.38–65.10 0.97–14.08 0.01–159.10 0.17–62.33 4.01–215.50
51.35 31.76 27.36 39.27 4.91 2.14 22.05 10.40 12.45
CVD, cardiovascular disease; OD, other diseases; P, poisoning; A, asphyxia; D, drowning; H, hypothermia; TT, thoracic trauma; OT, other trauma; F, fire fatality.
although Ellingsen and Hetland [16] report serum cTnT being stable in the first 3 days after death and Peter et al. [21] found no correlation between serum cTnT levels and autolysis time. Only a few reports exist on cTnT levels in case of PMI above 75 h [3,21]. In our routine forensic practice, PMI tends to be relatively long; therefore, cases with PMI up to 141 h were included in the current study. Our findings suggest that the time-dependent elevation in both serum and pericardial fluid cTnT levels continues after 75 h postmortem. It has been suggested that postmortem cTnT could be used to support the diagnosis of heart disease as the cause of death if other causes of myocardial damage can be excluded [12,13,16] and to differentiate drowning and fatal hypothermia from acute myocardial infarction and methamphetamine abuse [12]. According to our findings, serum cTnT may be used as a supportive tool in differentiating deaths caused by CVD from poisoning and pericardial fluid cTnT in deaths caused by CVD from OT, as well as CVD and OD from drowning and hypothermia. However, the variation in cTnT levels among individual cases is high and there is overlap between groups, which makes it difficult to apply the results on individual cases. As mentioned above, cTnT clinical reference values are not applicable for postmortem investigations [12,13,16]. In the current study, all cases in the CVD group presented serum cTnT levels equal to or higher than the clinical reference of 0.03 ng/ml. Ellingsen and Hetland [16] evaluated the serum cTnT level of 0.6 mg/l as the cut-off value between evident/possible cardiac deaths and negative controls. In our study, 8/10 cases in the CVD group showed serum cTnT value above 0.6 ng/ml and 49/91 cases in all other groups combined represented with cTnT levels below that level. Cut-off values have been suggested by Zhu et al. [12] as 0.2 ng/ml for serum from external iliac venous blood, 10 ng/ml for pericardial fluid in the early postmortem period (within 12 h) to differentiate between drowning and other groups, and 0.6 ng/ml for serum from external iliac venous blood and 100 ng/ml for pericardial fluid 12–48 h postmortem to differentiate between higher cTnT level groups (hyperthermia, fatal methamphetamine abuse, carbon monoxide poisoning, acute myocardial infarction) and intermediate groups (injuries, asphyxiation and fire fatalities). In our study, with PMI within 12 h, other causes of death besides Table 3 CVD comorbidity. CVD comorbidity
CVD+ CVD
n
36 65
Serum cTnT (ng/ml)
PF cTnT (ng/ml)
Range
Median
Range
Median
0.07–214.70 0.00–500.00
0.59 0.78
0.04–250.00 0.01–310.40
22.61 18.06
CVD+ cases with comorbid cardiovascular disease, CVD cardiovascular disease.
cases without comorbid
drowning presented 3/8 cases with serum cTnT values above the suggested cut-off and none (0/8) with pericardial fluid cTnT above the cut-off; there were no drowning cases with PMI of less than 12 h. For 12–48 h PMI, we found acute myocardial infarction (1/1) with both serum and pericardial fluid cTnT above the suggested cut-off levels of 0.6 ng/ml and 100 ng/ml, respectively, there were no carbon monoxide poisoning cases with PMI 12–48 h. Trauma groups (TT and OT combined) presented cTnT values below the suggested cut-off for serum in 6/10 and for pericardial fluid in 9/10 cases, and the respective number of cases for asphyxiation was 5/ 12 and 11/12. The role of cardiac contusion and thoracic injury in increased postmortem troponin levels has been evaluated by several authors with some controversy [12,21,24,25]. Many cardiovascular conditions, such as congestive heart failure, hypertension, cardiomyopathy, myocarditis, aortic valve disease and cerebrovascular disease, are known to result in elevated cTnT levels [3,12,13,16,18]. We did not notice significantly elevated cTnT levels in the thoracic trauma group compared to other causes of death in the present study, and comorbid cardiovascular disease also seemed to have no effect on postmortem cTnT levels. Elevated cTnT levels in postmortem serum from peripheral blood may depend on the survival period following the onset of myocardial injury [12,13], which was not taken into consideration in the current paper due to difficulties obtaining correct data. In conclusion, the results of the current study generally confirm previous findings on postmortem serum and pericardial fluid cTnT. Postmortem cTnT, in addition to other forensic evidence, may help to define cause of death; however, the postmortem timedependent elevation in cTnT levels has to be taken into consideration. References [1] M.D. Perez-Carceles, J. Noguera, J.L. Jimenez, P. Martinez, A. Luna, E. Osuna, Diagnostic efficacy of biochemical markers in diagnosis post-mortem of ischaemic heart disease, Forensic Sci. Int. 142 (2004) 1–7. [2] F. Martı´nez Dı´az, M. Rodrı´guez-Morlensı´n, M.D. Pe´rez-Ca´rceles, J. Noguera, A. Luna, E. Osuna, Biochemical analysis and immunohistochemical determination of cardiac troponin for the postmortem diagnosis of myocardial damage, Histol. Histopathol. 20 (2005) 475–481. [3] S.J. Davies, D.C. Gaze, P.O. Collinson, Investigation of cardiac troponins in postmortem subjects: comparing antemortem and postmortem levels, Am. J. Forensic Med. Pathol. 26 (2005) 213–215. [4] G.L. de la Grandmaison, Is there progress in the autopsy diagnosis of sudden unexpected death in adults? Forensic Sci. Int. 156 (2006) 138–144. [5] B.L. Zhu, T. Ishikawa, T. Michiue, D.R. Li, D. Zhao, Y. Bessho, Y. Kamikodai, K. Tsuda, S. Okazaki, H. Maeda, Postmortem cardiac troponin I and creatine kinase MB levels in the blood and pericardial fluid as markers of myocardial damage in medicolegal autopsy, Leg. Med. 9 (2007) 241–250. [6] B.L. Zhu, T. Ishikawa, T. Michiue, D.R. Li, D. Zhao, S. Tanaka, Y. Kamikodai, K. Tsuda, S. Okazaki, H. Maeda, Postmortem pericardial natriuretic peptides as markers of cardiac function in medico-legal autopsies, Int. J. Legal. Med. 121 (2007) 28–35. [7] H. Maeda, B.L. Zhu, T. Ishikawa, L. Quan, T. Michiue, Significance of postmortem biochemistry in determining the cause of death, Leg. Med. 11 (2009) S46–S49. [8] A. Luna, Is postmortem biochemistry really useful? Why is not widely used in forensic pathology?, Leg. Med. 11 (2009) S27–S30. [9] Q. Wang, T. Michiue, T. Ishikawa, B.L. Zhu, H. Maeda, Combined analyses of creatine kinase MB, cardiac troponin I and myoglobin in pericardial and cerebrospinal fluids to investigate myocardial and skeletal muscle injury in medicolegal autopsy cases, Leg. Med. 13 (2011) 226–232. [10] C. Palmiere, P. Mangin, Postmortem chemistry update part II, Int. J. Legal Med. 126 (2012) 199–215. [11] C. Palmiere, et al., Biochemical markers of fatal hypothermia, Forensic Sci. Int. 226 (2013) 54–61. [12] B.L. Zhu, T. Ishikawa, T. Michiue, D.R. Li, D. Zhao, S. Oritani, Y. Kamikodai, K. Tsuda, S. Okazaki, H. Maeda, Postmortem cardiac troponin T levels in the blood and pericardial fluid. Part 1: analysis with special regard to traumatic causes of death, Leg. Med. 8 (2006) 86–93. [13] B.L. Zhu, T. Ishikawa, T. Michiue, D.R. Li, D. Zhao, Y. Kamikodai, K. Tsuda, S. Okazaki, H. Maeda, Postmortem cardiac troponin T levels in the blood and pericardial fluid. Part 2: analysis for application in the diagnosis of sudden cardiac death with regard to pathology, Leg. Med. 8 (2006) 94–101. [14] H. Maeda, T. Michiue, B.L. Zhu, T. Ishikawa, L. Quan, Analysis of cardiac troponins and creatine kinase MB in cerebrospinal fluid in medicolegal autopsy cases, Leg. Med. 11 (2009) S266–S268.
S. Remmer et al. / Forensic Science International 233 (2013) 154–157 [15] A.B. Khalifa, M. Najjar, F. Addad, E. Turki, T. Mghirbi, Cardiac troponin T (cTnT) and the postmortem diagnosis of sudden death, Am. J. Forensic Med. Pathol. 27 (2006) 175–177. [16] C.L. Ellingsen, Ø. Hetland, Serum concentrations of cardiac troponin T in sudden death, Am. J. Forensic Med. Pathol. 25 (2004) 213–215. [17] B.L. Zhu, K. Ishida, M. Taniguchi, L. Quan, S. Oritani, K. Tsuda, Y. Kamikodai, M.Q. Fujita, H. Maeda, Possible postmortem serum markers for differentiation between fresh-, saltwater drowning and acute cardiac death: a preliminary investigation, Leg. Med. 5 (2003) S298–S301. [18] A.S. Jaffe, L. Babuin, F.S. Apple, Biomarkers in acute cardiac disease: the present and the future, J. Am. Coll. Cardiol. 48 (2006) 1–11. [19] B. Madea, F. Musshoff, Postmortem biochemistry, Forensic Sci. Int. 165 (2007) 165–171. [20] H. Maeda, T. Ishikawa, T. Michiue, Forensic biochemistry for functional investigation of death: concept and practical application, Leg. Med. 13 (2011) 55–67.
157
[21] J. Peter, A. Kirchner, E. Kuhlisch, M. Menschikowski, B. Neef, J. Dressler, The relevance of the detection of troponins to the forensic diagnosis of cardiac contusion, Forensic Sci. Int. 160 (2006) 127–133. [22] A. Thierauf, F. Musshoff, B. Madea, Post-mortem biochemical investigations of vitreous humor, Forensic Sci. Int. 192 (2009) 78–82. [23] J.I. Coe, Postmortem chemistry update, Am. J. Forensic Med. Pathol. 14 (1993) 91–117. [24] S.J. Cina, W.C. Thompson, R.J. Fischer, D.K. Brown, J.M. Titus, J.E. Smialek, A study of various morphologic variables and troponin I in pericardial fluid as possible discriminators of sudden cardiac death, Am. J. Forensic Med. Pathol. 20 (1999) 333–337. [25] S.J. Cina, D.J. Li, D.W. Chan, J.K. Boitnott, R.H. Hruban, J.E. Smialek, Serum concentrations of cardiac troponin I in sudden death: a pilot study, Am. J. Forensic Med. Pathol. 19 (1998) 324–328.
Contents lists available at ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Cardiac troponin T in forensic autopsy cases S. Remmer a,b,*, A. Kuudeberg a, M. To˜nisson a, D. Lepik a, M. Va¨li a,b a b
Institute of Pathological Anatomy and Forensic Medicine, University of Tartu, Ravila 19, 50411 Tartu, Estonia Estonian Forensic Science Institute, Tervise 30, 13419 Tallinn, Estonia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 2 May 2013 Received in revised form 4 September 2013 Accepted 6 September 2013 Available online 14 September 2013
The aim of the present study was to describe the findings of postmortem serum and pericardial fluid (PF) cardiac troponin T (cTnT) in various causes of death with regard to the postmortem interval (PMI) and comorbid cardiovascular disease, using 101 autopsy cases with PMI of 8–141 h divided into 9 groups: cardiovascular disease (CVD), other diseases (OD), poisoning (P), asphyxia (A), drowning (D), hypothermia (H), thoracic trauma (TT), other trauma (OT) and fire fatalities (F). The results suggest that cTnT levels may help to differentiate cardiovascular death from poisoning and non-thoracic trauma, as well as to differentiate cardiovascular and other diseases as cause of death from drowning and hypothermia. However, the effect of PMI, unlike comorbid cardiovascular disease, has to be taken into account. ß 2013 Elsevier Ireland Ltd. All rights reserved.
Keywords: Postmortem biochemistry Cardiac troponin T Blood Pericardial fluid
1. Introduction The use of cardiac troponins as a part of laboratory investigations in forensic autopsy has been analysed by several authors [1– 11]. Cardiac troponin T (cTnT), a marker for myocardial injury, has been recommended for the evaluation of the presence and extent of myocardial damage in various causes of death [12–15]. cTnT may be used to confirm the diagnosis of cardiac-related death and in distinguishing certain causes of deaths from others [12,13,15– 17]. In the living, an increase in the level of cTnT in serum can be seen approximately 2–4 h after the onset of cardiac symptoms and persists for up to 2 weeks [18]. Postmortem cTnT levels depend on several factors, such as the severity and duration of myocardial damage before death, the cause of death, the postmortem interval (the time between death and the collecting of samples, PMI) and the sampling site [6,8,9,12–14,19–21]; therefore, clinical serum cTnT reference values cannot be directly used in interpreting postmortem samples and cut-off values for postmortem cTnT levels have been suggested [12,13,16]. Besides acute myocardial infarction, other conditions, especially cardiovascular diseases, are known to result in elevated cTnT levels [3,12,13,16,18], and the effect of comorbid disease on postmortem cTnT levels should be
* Corresponding author at: Institute of Pathological Anatomy and Forensic Medicine, University of Tartu, Ravila 19, 50411 Tartu, Estonia. Tel.: +372 7374290. E-mail addresses: [email protected] (S. Remmer), [email protected] (A. Kuudeberg), [email protected] (M. To˜nisson), [email protected] (D. Lepik), [email protected] (M. Va¨li). 0379-0738/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.forsciint.2013.09.010
taken into account. Due to rapid postmortem putrefaction of blood, the use of alternative biological fluids, such as pericardial fluid (PF) is advised [8,19,20,22,23]. There is no previous experience using postmortem cardiac markers as a diagnostic tool in forensic autopsies in Estonia. The aim of this study is to describe the findings regarding the possible use of postmortem serum and pericardial fluid cTnT in investigating the cause of death. 2. Materials and methods 2.1. Materials A total of 230 forensic autopsy cases at the Estonian Forensic Science Institute were examined. At autopsy, blood was drawn from femoral/iliac veins and pericardial fluid was collected after opening the pericardial sac. PF samples contaminated with blood and all cases with evident decomposition were excluded. The final sample consisted of 101 cases (87 males and 14 females), 25– 54 years of age with a median of 42 years of age. The estimated postmortem interval was 8–141 h with a median of 34.5 h. The cause of death was grouped on the basis of gross, histological and toxicologial findings as follows: cardiovascular disease (CVD, n = 10), other diseases (OD, n = 12), poisoning (P, n = 20), asphyxia (A, n = 25), drowning (D, n = 11), hypothermia (H, n = 5), blunt and sharp thoracic trauma (TT, n = 7), other trauma (OT, n = 8) and fire fatalities (carbon monoxide poisoning, F, n = 3). The CVD group included cases with International Classification of Diseases codes I20–25 and I 30-52. The poisoning group consisted of cases with acute lethal poisoning by the following agents: ethanol, surrogate alcohol, diethyl ether, diazepam, phenobarbital, pentobarbital, thiopental, clozapine, tramadol, fentanyl, 3-methylfentanyl, methadone and amphetamine. All cases were classified according to the presence or absence of comorbid chronic cardiovascular disease (n = 36 and n = 65, respectively). PMI was divided into 5 groups: less than 12 h (n = 8), 12–24 h (n = 20), 24–48 h (n = 41), 48–72 h (n = 21) and over 72 h (n = 11). Case profiles are shown in Table 1. Due to the small number of cases, fire fatalities were left out of further statistical analysis.
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Table 1 Case profiles (n = 101, 87 male and 14 female). Cause of death
n
Age (years)
PMI (h)
Range
Median
Range
Median
CVD OD P A D H TT OT F
10 12 20 25 11 5 7 8 3
28–53 30–54 27–52 41–54 25–51 27–54 26–54 29–54 43–51
48.0 42.5 40.0 40.0 39.0 48.0 37.0 42.5 48.0
16.5–100.0 11.0–129.5 9.0–84.0 8.0–96.0 13.5–57.5 22.0–106.0 8.5–66.3 8.0–74.0 10.5–141.0
34.3 34.0 35.0 36.0 35.5 35.0 16.0 25.8 106.0
CVD, cardiovascular disease; OD, other diseases; P, poisoning; A, asphyxia; D, drowning; H, hypothermia; TT, thoracic trauma; OT, other trauma; F, fire fatality; PMI, postmortem interval (h) hours.
2.2. Biochemical analyses Samples were stored at +4 8C during transportation to the laboratory (24 h). Blood was centrifuged to separate the serum. Samples were stored at 20 8C until use. cTnT was measured using electro-chemiluminescence immunoassay (Roche Diagnostics Elecsys). The upper limit for clinical reference range was 0.03 ng/ml. 2.3. Statistical methods Spearman’s rank order correlation (rs) was used to evaluate the relationship between pairs of parameters. Non-parametric Kruskal–Wallis test was used to compare groups and the non-parametric Mann–Whitney U test was used for comparisons between individual groups. Statistical analysis was performed using statistical software R (version 2.14.0) and Statistica 12.0. A p-value of less than 0.05 was considered statistically significant.
3. Results We found no significant relationship between cTnT levels and age or gender. There was a positive correlation between serum cTnT and pericardial fluid cTnT levels (rs = 0.43, p < 0.001, Spearman). The level of pericardial fluid cTnT was higher compared to serum cTnT among all groups of causes of death, except for asphyxiation and hypothermia. When compared pairwise, pericardial fluid cTnT levels were higher compared to serum cTnT levels in most cases (90/101) and equal in 1 case. In cases with higher serum cTnT compared to pericardial fluid cTnT, the cause of death was asphyxiation in 5 out of 10. We found a moderate positive correlation between pericardial fluid cTnT level and PMI (rs = 0.41, p < 0.001, Spearman) and a weak positive correlation between serum cTnT and PMI (rs = 0.22, p = 0.02, Spearman). A significant postmortem time-dependent elevation in serum and pericardial fluid cTnT levels emerged between different PMI groups (p-value = 0.04 for serum and pvalue = 0.004 for pericadial fluid, Kruskal–Wallis, Figs. 1 and 2). Among different groups of COD, postmortem time-dependent elevation in pericardial fluid cTnT levels was found for asphyxiation (rs = 0.80, p-value < 0.001, Spearman). No statistically important postmortem elevation in serum cTnT levels for COD groups was noticed.cTnT levels in serum and pericardial fluid regarding different causes of death are shown in Table 2. Serum cTnT levels in the CVD group were significantly higher compared to poisoning (pvalue < 0.01, Mann–Whitney U test). Pericardial fluid cTnT levels showed significantly higher results in the CVD group compared to drowning (p-value < 0.01), other trauma (p-value = 0.03) and hypothermia (p-value < 0.01). Also, pericardial fluid cTnT levels were significantly higher in OD group compared to drowning (pvalue = 0.02) and hypothermia (p-value = 0.03). We found no significant difference in the levels of serum and PF cTnT between cases with and without CVD comorbidity for all COD
Fig. 1. Serum cTnT levels according to PMI interval.
(p-value = 0.7549 for serum, p-value = 0.3402 for pericardial fluid, Kruskal–Wallis) or among individual COD groups (p-value = 0.7575 and p-value = 0.342 respectively, Mann–Whitney Utest, Table 3).
4. Discussion Cardiac troponin T (cTnT) has previously been assessed for the postmortem diagnosis of myocardial injury with some controversy. Postmortem cTnT levels are generally lower in serum from peripheral blood compared to the levels in pericardial fluid, and the proximity of the myocardium and direct leakage of cTnT into the pericardium has been suggested as the cause [12,13,24]. Correspondingly, pericardial fluid cTnT levels were generally higher compared to serum cTnT levels in our study. We found a positive correlation between the cTnT levels in serum and pericardial fluid, which has not been previously reported. A postmortem time-dependent elevation in cTnT levels in the first days after death has been shown previously [6,9,12,13],
Fig. 2. Pericardial fluid cTnT levels according to PMI interval.
S. Remmer et al. / Forensic Science International 233 (2013) 154–157
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Table 2 Serum and pericardial fluid cTnT regarding cause of death. Cause of death
n
CVD OD P A D H TT OT F
10 12 20 25 11 5 7 8 3
Serum cTnT (ng/ml)
PF cTnT (ng/ml)
Range
Median
Range
Median
0.03–84.36 0.01–214.70 0.01–80.96 0.01–500.00 0.01–17.54 0.01–24.02 0.01–39.28 0.01–13.66 0.01–0.81
10.56 1.10 0.21 0.60 0.83 0.28 0.20 0.55 0.06
4.98–250.00 0.51–310.40 0.04–192.70 0.19–271.90 0.38–65.10 0.97–14.08 0.01–159.10 0.17–62.33 4.01–215.50
51.35 31.76 27.36 39.27 4.91 2.14 22.05 10.40 12.45
CVD, cardiovascular disease; OD, other diseases; P, poisoning; A, asphyxia; D, drowning; H, hypothermia; TT, thoracic trauma; OT, other trauma; F, fire fatality.
although Ellingsen and Hetland [16] report serum cTnT being stable in the first 3 days after death and Peter et al. [21] found no correlation between serum cTnT levels and autolysis time. Only a few reports exist on cTnT levels in case of PMI above 75 h [3,21]. In our routine forensic practice, PMI tends to be relatively long; therefore, cases with PMI up to 141 h were included in the current study. Our findings suggest that the time-dependent elevation in both serum and pericardial fluid cTnT levels continues after 75 h postmortem. It has been suggested that postmortem cTnT could be used to support the diagnosis of heart disease as the cause of death if other causes of myocardial damage can be excluded [12,13,16] and to differentiate drowning and fatal hypothermia from acute myocardial infarction and methamphetamine abuse [12]. According to our findings, serum cTnT may be used as a supportive tool in differentiating deaths caused by CVD from poisoning and pericardial fluid cTnT in deaths caused by CVD from OT, as well as CVD and OD from drowning and hypothermia. However, the variation in cTnT levels among individual cases is high and there is overlap between groups, which makes it difficult to apply the results on individual cases. As mentioned above, cTnT clinical reference values are not applicable for postmortem investigations [12,13,16]. In the current study, all cases in the CVD group presented serum cTnT levels equal to or higher than the clinical reference of 0.03 ng/ml. Ellingsen and Hetland [16] evaluated the serum cTnT level of 0.6 mg/l as the cut-off value between evident/possible cardiac deaths and negative controls. In our study, 8/10 cases in the CVD group showed serum cTnT value above 0.6 ng/ml and 49/91 cases in all other groups combined represented with cTnT levels below that level. Cut-off values have been suggested by Zhu et al. [12] as 0.2 ng/ml for serum from external iliac venous blood, 10 ng/ml for pericardial fluid in the early postmortem period (within 12 h) to differentiate between drowning and other groups, and 0.6 ng/ml for serum from external iliac venous blood and 100 ng/ml for pericardial fluid 12–48 h postmortem to differentiate between higher cTnT level groups (hyperthermia, fatal methamphetamine abuse, carbon monoxide poisoning, acute myocardial infarction) and intermediate groups (injuries, asphyxiation and fire fatalities). In our study, with PMI within 12 h, other causes of death besides Table 3 CVD comorbidity. CVD comorbidity
CVD+ CVD
n
36 65
Serum cTnT (ng/ml)
PF cTnT (ng/ml)
Range
Median
Range
Median
0.07–214.70 0.00–500.00
0.59 0.78
0.04–250.00 0.01–310.40
22.61 18.06
CVD+ cases with comorbid cardiovascular disease, CVD cardiovascular disease.
cases without comorbid
drowning presented 3/8 cases with serum cTnT values above the suggested cut-off and none (0/8) with pericardial fluid cTnT above the cut-off; there were no drowning cases with PMI of less than 12 h. For 12–48 h PMI, we found acute myocardial infarction (1/1) with both serum and pericardial fluid cTnT above the suggested cut-off levels of 0.6 ng/ml and 100 ng/ml, respectively, there were no carbon monoxide poisoning cases with PMI 12–48 h. Trauma groups (TT and OT combined) presented cTnT values below the suggested cut-off for serum in 6/10 and for pericardial fluid in 9/10 cases, and the respective number of cases for asphyxiation was 5/ 12 and 11/12. The role of cardiac contusion and thoracic injury in increased postmortem troponin levels has been evaluated by several authors with some controversy [12,21,24,25]. Many cardiovascular conditions, such as congestive heart failure, hypertension, cardiomyopathy, myocarditis, aortic valve disease and cerebrovascular disease, are known to result in elevated cTnT levels [3,12,13,16,18]. We did not notice significantly elevated cTnT levels in the thoracic trauma group compared to other causes of death in the present study, and comorbid cardiovascular disease also seemed to have no effect on postmortem cTnT levels. Elevated cTnT levels in postmortem serum from peripheral blood may depend on the survival period following the onset of myocardial injury [12,13], which was not taken into consideration in the current paper due to difficulties obtaining correct data. In conclusion, the results of the current study generally confirm previous findings on postmortem serum and pericardial fluid cTnT. Postmortem cTnT, in addition to other forensic evidence, may help to define cause of death; however, the postmortem timedependent elevation in cTnT levels has to be taken into consideration. References [1] M.D. Perez-Carceles, J. Noguera, J.L. Jimenez, P. Martinez, A. Luna, E. Osuna, Diagnostic efficacy of biochemical markers in diagnosis post-mortem of ischaemic heart disease, Forensic Sci. Int. 142 (2004) 1–7. [2] F. Martı´nez Dı´az, M. Rodrı´guez-Morlensı´n, M.D. Pe´rez-Ca´rceles, J. Noguera, A. Luna, E. Osuna, Biochemical analysis and immunohistochemical determination of cardiac troponin for the postmortem diagnosis of myocardial damage, Histol. Histopathol. 20 (2005) 475–481. [3] S.J. Davies, D.C. Gaze, P.O. Collinson, Investigation of cardiac troponins in postmortem subjects: comparing antemortem and postmortem levels, Am. J. Forensic Med. Pathol. 26 (2005) 213–215. [4] G.L. de la Grandmaison, Is there progress in the autopsy diagnosis of sudden unexpected death in adults? Forensic Sci. Int. 156 (2006) 138–144. [5] B.L. Zhu, T. Ishikawa, T. Michiue, D.R. Li, D. Zhao, Y. Bessho, Y. Kamikodai, K. Tsuda, S. Okazaki, H. Maeda, Postmortem cardiac troponin I and creatine kinase MB levels in the blood and pericardial fluid as markers of myocardial damage in medicolegal autopsy, Leg. Med. 9 (2007) 241–250. [6] B.L. Zhu, T. Ishikawa, T. Michiue, D.R. Li, D. Zhao, S. Tanaka, Y. Kamikodai, K. Tsuda, S. Okazaki, H. Maeda, Postmortem pericardial natriuretic peptides as markers of cardiac function in medico-legal autopsies, Int. J. Legal. Med. 121 (2007) 28–35. [7] H. Maeda, B.L. Zhu, T. Ishikawa, L. Quan, T. Michiue, Significance of postmortem biochemistry in determining the cause of death, Leg. Med. 11 (2009) S46–S49. [8] A. Luna, Is postmortem biochemistry really useful? Why is not widely used in forensic pathology?, Leg. Med. 11 (2009) S27–S30. [9] Q. Wang, T. Michiue, T. Ishikawa, B.L. Zhu, H. Maeda, Combined analyses of creatine kinase MB, cardiac troponin I and myoglobin in pericardial and cerebrospinal fluids to investigate myocardial and skeletal muscle injury in medicolegal autopsy cases, Leg. Med. 13 (2011) 226–232. [10] C. Palmiere, P. Mangin, Postmortem chemistry update part II, Int. J. Legal Med. 126 (2012) 199–215. [11] C. Palmiere, et al., Biochemical markers of fatal hypothermia, Forensic Sci. Int. 226 (2013) 54–61. [12] B.L. Zhu, T. Ishikawa, T. Michiue, D.R. Li, D. Zhao, S. Oritani, Y. Kamikodai, K. Tsuda, S. Okazaki, H. Maeda, Postmortem cardiac troponin T levels in the blood and pericardial fluid. Part 1: analysis with special regard to traumatic causes of death, Leg. Med. 8 (2006) 86–93. [13] B.L. Zhu, T. Ishikawa, T. Michiue, D.R. Li, D. Zhao, Y. Kamikodai, K. Tsuda, S. Okazaki, H. Maeda, Postmortem cardiac troponin T levels in the blood and pericardial fluid. Part 2: analysis for application in the diagnosis of sudden cardiac death with regard to pathology, Leg. Med. 8 (2006) 94–101. [14] H. Maeda, T. Michiue, B.L. Zhu, T. Ishikawa, L. Quan, Analysis of cardiac troponins and creatine kinase MB in cerebrospinal fluid in medicolegal autopsy cases, Leg. Med. 11 (2009) S266–S268.
S. Remmer et al. / Forensic Science International 233 (2013) 154–157 [15] A.B. Khalifa, M. Najjar, F. Addad, E. Turki, T. Mghirbi, Cardiac troponin T (cTnT) and the postmortem diagnosis of sudden death, Am. J. Forensic Med. Pathol. 27 (2006) 175–177. [16] C.L. Ellingsen, Ø. Hetland, Serum concentrations of cardiac troponin T in sudden death, Am. J. Forensic Med. Pathol. 25 (2004) 213–215. [17] B.L. Zhu, K. Ishida, M. Taniguchi, L. Quan, S. Oritani, K. Tsuda, Y. Kamikodai, M.Q. Fujita, H. Maeda, Possible postmortem serum markers for differentiation between fresh-, saltwater drowning and acute cardiac death: a preliminary investigation, Leg. Med. 5 (2003) S298–S301. [18] A.S. Jaffe, L. Babuin, F.S. Apple, Biomarkers in acute cardiac disease: the present and the future, J. Am. Coll. Cardiol. 48 (2006) 1–11. [19] B. Madea, F. Musshoff, Postmortem biochemistry, Forensic Sci. Int. 165 (2007) 165–171. [20] H. Maeda, T. Ishikawa, T. Michiue, Forensic biochemistry for functional investigation of death: concept and practical application, Leg. Med. 13 (2011) 55–67.
157
[21] J. Peter, A. Kirchner, E. Kuhlisch, M. Menschikowski, B. Neef, J. Dressler, The relevance of the detection of troponins to the forensic diagnosis of cardiac contusion, Forensic Sci. Int. 160 (2006) 127–133. [22] A. Thierauf, F. Musshoff, B. Madea, Post-mortem biochemical investigations of vitreous humor, Forensic Sci. Int. 192 (2009) 78–82. [23] J.I. Coe, Postmortem chemistry update, Am. J. Forensic Med. Pathol. 14 (1993) 91–117. [24] S.J. Cina, W.C. Thompson, R.J. Fischer, D.K. Brown, J.M. Titus, J.E. Smialek, A study of various morphologic variables and troponin I in pericardial fluid as possible discriminators of sudden cardiac death, Am. J. Forensic Med. Pathol. 20 (1999) 333–337. [25] S.J. Cina, D.J. Li, D.W. Chan, J.K. Boitnott, R.H. Hruban, J.E. Smialek, Serum concentrations of cardiac troponin I in sudden death: a pilot study, Am. J. Forensic Med. Pathol. 19 (1998) 324–328.
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