Journal of Forensic Science and Medicine

ORIGINAL ARTICLE
Year
: 2022  |  Volume : 8  |  Issue : 4  |  Page : 135--141

Analyses of N-Terminal pro-brain natriuretic peptide, cardiac troponin T, and creatine kinase MB in pericardial fluid in sudden cardiac death caused by ischemic heart disease


Zhipeng Cao, Tianqi Wang, Shao-Huang Wu, Zihan Liao, Baoli Zhu, Rui Zhao 
 Department of Forensic Pathology, School of Forensic Medicine, China Medical University; Liaoning Province Key Laboratory of Forensic Bio-Evidence Sciences; Center of Forensic Investigation, China Medical University, Shenyang, China

Correspondence Address:
Prof. Rui Zhao
Department of Forensic Pathology, School of Forensic Medicine, China Medical University; Liaoning Province Key Laboratory of Forensic Bio-Evidence Sciences; Center of Forensic Investigation, China Medical University, Shenyang
China

Abstract

Background: Biochemical analyses of N-terminal pro-brain natriuretic peptide (NT-proBNP), cardiac troponin T (cTnT), and creatine kinase MB (CK-MB) have been reported to be valuable for the auxiliary diagnosis of sudden cardiac death (SCD) in previous forensic studies. Aims and Objectives: The present study aimed to evaluate the diagnostic efficiency of combined analyses of NT-proBNP, cTnT and CK-MB in the pericardial fluid for forensic diagnosis of SCD caused by ischemic heart disease. Materials and Methods: Levels of NT-proBNP, cTnT, and CK-MB in the pericardial fluid of 132 medicolegal autopsy cases were obtained through electrochemiluminescence method. Results: NT-proBNP, cTnT, and CK-MB levels were significantly elevated in SCD cases (P < 0.05). Receiver-operating characteristics (ROC) analysis showed that NT-proBNP, cTnT, and CK-MB have diagnostic value for the diagnosis of SCD: NT-proBNP, cutoff value of 2236 pg/ml; cTnT, cutoff value of 199.51 ng/ml; CK-MB: cutoff value of 2742.5 ng/ml, and the combined analyses of these three biomarkers have better diagnostic efficiency than each single biomarker alone. Moreover, the causes of SCD were sub-divided into acute ischemic heart disease, acute myocardial infarction (AMI), and recurrent myocardial infarction subgroups for further analysis, which revealed that the ratio of cTnT/CK-MB could be used to distinguish AMI with the cutoff value of 0.1085 estimated by ROC analysis. Conclusion: These observations suggested that the postmortem biochemical analyses of NT-proBNP, cTnT, and CK-MB in the pericardial fluid may assist to diagnose SCD in forensic practice, and the combined analyses of multiple biomarkers have better diagnostic efficiency than each single biomarker alone. On the basis of the postmortem biochemical analyses of NT-proBNP, cTnT and CK-MB, combining the ratio of cTnT/CK-MB could be used to distinguish AMI.



How to cite this article:
Cao Z, Wang T, Wu SH, Liao Z, Zhu B, Zhao R. Analyses of N-Terminal pro-brain natriuretic peptide, cardiac troponin T, and creatine kinase MB in pericardial fluid in sudden cardiac death caused by ischemic heart disease.J Forensic Sci Med 2022;8:135-141


How to cite this URL:
Cao Z, Wang T, Wu SH, Liao Z, Zhu B, Zhao R. Analyses of N-Terminal pro-brain natriuretic peptide, cardiac troponin T, and creatine kinase MB in pericardial fluid in sudden cardiac death caused by ischemic heart disease. J Forensic Sci Med [serial online] 2022 [cited 2023 Jan 28 ];8:135-141
Available from: https://www.jfsmonline.com/text.asp?2022/8/4/135/366417


Full Text

 Introduction



Sudden cardiac death (SCD), as a death due to sudden loss of cardiac function, has seriously threatened lives. There are seven million people die from SCD each year around the world.[1],[2],[3] With high morbidity and mortality, the postmortem diagnosis of SCD has become one of the common causes in routine forensic practice.[4] However, previous studies showed that there were about 5%–10% of SCD cases lacking of typical morphological changes through routine autopsy and histological examinations, and even 1%–5% of SCD cases revealed negative autopsy.[5],[6] Therefore, it is necessary to employ various auxiliary investigation methods for comprehensive and accurate diagnosis of the cause of death in suspected SCD.

In the clinical diagnosis of heart diseases, N-terminal pro-brain natriuretic peptide (NT-proBNP), cardiac troponin T/I (cTnT/I), and creatine kinase MB (CK-MB) are always used as classical biomarkers for evaluating the cardiac function and myocardial injury; they provide useful diagnostic basis for physicians.[7],[8],[9] In forensic studies, postmortem biochemical analyses of NT-proBNP, cTnT/I, and CK-MB in the postmortem specimen have also been reported to be valuable for the evaluation of cardiac function and myocardial injury, and they were recommended as auxiliary and valuable biomarkers for the forensic diagnosis of SCD.[10],[11]

Although previous studies have demonstrated the application of NT-proBNP, cTnT/I, and CK-MB in forensic practice, there is no report on the value and efficiency of combined postmortem biochemical analyses of NT-proBNP, cTnT/I, and CK-MB in forensic diagnosis of SCD.[12],[13],[14] As a result, the present study investigated the levels of NT-proBNP, cTnT, and CK-MB in the pericardial fluid of SCD cases and control cases and further evaluated the diagnostic efficiency of these three biomarkers in the forensic diagnosis of SCD.

 Materials and Methods



Sample collection

Medicolegal autopsy cases from 2020 to 2022 at Center of Forensic Investigation, China Medical University (n = 132) were included. The cadavers included in the study were either autopsied within 48 h after death or placed in cryopreservation at - 2°C within 12 h after death and autopsied within 7 days to exclude decomposed specimens. These cases comprised 87 males and 45 females, 16–89 years of age. Since pericardial fluid is similar to plasma in composition and exists in a closed pericardial cavity, it is less affected by postmortem changes. Therefore, the present study used pericardial fluid as the test material for the examination and analysis. The pericardial fluid was collected with disposable Pap straws after the pericardial cavity was opened during autopsy and stored in a 2 ml sterile centrifuge tube, and was tested after centrifuging at 3000 g for 15 min. The samples that were not tested on time were frozen at − 80°C for biochemical analysis tested.

Group

The causes of the death of the 132 cases that involved in the present study were concluded by at least three independent forensic pathologists, certified by Liaoning Provincial Department of Justice, based on comprehensive autopsy, histological examination, and toxicological analysis. The cases were divided into SCD group and control group according to the causes of death as follows: SCD groups (n = 107, comprising 71 males and 31 females, 20–89 years of age) consisting of acute ischemic heart disease (AIHD, n = 48, comprising 34 males and 14 females, 20–71 years of age), acute myocardial infarction (AMI, n = 19, comprising 15 males and 4 females, 28–89 years of age), recurrent myocardial infarction (RMI, n = 35, comprising 23 males and 12 females, 33–73 years of age); control group (Con, n = 30, comprising 16 males and 14 females, 16–85 years of age). According to previous reports, cases involving complication that may have influenced the pathophysiological process of death such as liver cirrhosis, renal failure, and intoxication were excluded in the cases mentioned above, and cardiopulmonary diseases such as coronary artery with over Grade II stenosis, cardiomyopathy, pneumonia and pulmonary fibrosis, and cardiopulmonary resuscitation acceptors were excluded in the control group.[15]

Biochemical analysis

NT-proBNP, cTnT, and CK-MB in pericardial fluid were analyzed using the electrochemiluminescence method and performed by Roche cobas e411 (Roche Diagnostic GmbH, Mannheim Germany) as an automatic electrochemiluminescence-analyzer. The value of NT-proBNP, cTnT, and CK-MB that exceeded the detection limit of the assay kit was recorded as the upper limit: NT-proBNP was recorded as 35000 pg/ml; cTnT was recorded as 1000 ng/ml; CK-MB was recorded as 30000 ng/ml.

Statistical analysis

The comparisons between the groups were performed using the Mann–Whitney U-test. The sensitivity and specificity in distinguishing the two groups using a cutoff value for each biomarker were estimated by Receiver-operating characteristics (ROC) analysis was used to perform the analysis of diagnostic efficiency and determination of cutoff value. The SPSS 20.0 (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis mentioned above, and GraphPad Prism 8.0 (GraphPad, San Diego, California, USA) was used for production of graphs. P < 0.05 was considered statistically significant.

Ethical approval

This study was performed in accordance with the 1964 Declaration of Helsinki and was approved by Ethics Committee of China Medical University in November, 2019 (CMU201901961). Biological samples were taken with the consent of the deceased's family and informed consent was signed with the family. All biological samples were anonymized before analysis and analyzed in the same laboratory.

 Results



N-terminal pro-brain natriuretic peptide, cardiac troponin T, and creatine kinase MB in the pericardial fluid of the sudden cardiac death group

Compared with the control group, the levels of NT-proBNP, cTnT, and CK-MB in the pericardial fluid of the SCD group were statistically elevated [Table 1]. ROC analysis showed that NT-proBNP, cTnT, and CK-MB had diagnostic significance (P < 0.05), and the cutoff values for NT-proBNP, cTnT, and CK-MB were 2236 pg/ml, 199.51 ng/ml, and 2742.5 ng/ml, respectively [Table 2] and [Figure 1]. Additionally, combined analyses (area under the curve [AUC] = 0.912) of NT-proBNP, cTnT, and CK-MB had better diagnostic efficiency than the each single biomarker alone (AUC of single NT-proBNP, cTnT, and CK-MB was 0.653, 0.844, and 0.808, respectively).{Table 1}{Table 2}{Figure 1}

N-terminal pro-brain natriuretic peptide, cardiac troponin T, and creatine kinase MB in the pericardial fluid of acute ischemic heart disease, acute myocardial infarction, and recurrent myocardial infarction subgroups

As SCD groups were further sub-divided into three subgroups, including AIHD, AMI, and RMI, further analyses showed that the levels of NT-proBNP in AMI and RMI subgroup were significantly higher than those in the control group and the AIHD subgroup (P < 0.05); the levels of cTnT and CK-MB in AIHD, AMI and RMI were significantly higher than those in the control group (P < 0.05); and levels of CK-MB in AIHD and RMI were significantly higher than those in AMI subgroup [P < 0.05, [Table 3] and [Figure 2]].{Table 3}{Figure 2}

The ratio of cardiac troponin T/creatine kinase MB in acute myocardial infarction, acute ischemic heart disease, and recurrent myocardial infarction subgroups

The cTnT/CK-MB ratio in AMI subgroup was significantly higher than those in AIHD, RMI subgroups and the control group (P < 0.05). As a result, AIHD and RMI subgroups were merged to perform ROC analysis, which showed that cTnT/CK-MB ratio contributed to distinguish AMI subgroup from AIHD and RMI subgroups with a cutoff value of 0.1085 [Figure 3].{Figure 3}

Analysis of representative cases

In order to facilitate the practical application of the above results, we selected one representative case from the AIHD, AMI, RMI subgroups, and control group, respectively, and listed levels of NT-proBNP, cTnT, CK-MB, and cTnT/CK-MB in the pericardial fluid [Table 4]. As shown in the table, cTnT and CK-MB levels in pericardial fluid in Case 1 (AIHD), Case 2 (AMI) and Case 3 (RMI) were higher than those in Case 4 (control), and CK-MB levels in Case 1 and Case 3 were higher than those in Case 2. As for NT-proBNP, Case 2 and Case 3 showed higher NT-proBNP levels than those in Case 4, which was not observed in Case 1. The cTnT/CK-MB ratio in Case 2 was higher than that in Case 1 and Case 3.{Table 4}

 Discussion



NT-proBNP, cTnT, and CK-MB, as crucial indicators for evaluating cardiac function and myocardial injury in clinical practice, have been demonstrated to be valuable for postmortem diagnosis of SCD in recent forensic studies.[10],[11] Similar to clinical practice, NT-proBNP in pericardial fluid is a valuable indicator for postmortem assessment of cardiac function;[4],[16] cTnT and CK-MB take the supporting role of diagnosis of myocardial injury,[17],[18],[19],[20] which confirmed the availability of NT-proBNP, cTnT, and CK-MB as auxiliary indicators for the diagnosis of SCD.

Previous postmortem biochemistry studies have shown that PMI is an important factor influencing the biochemical analysis. Palmiere et al. divided case with different PMI into 4–24 h PMI group, 25–48 h PMI group and 49–72 h PMI group and demonstrated that no statistical difference was found in cTnI, cTnT, and NT-proBNP in the 4–24 h PMI and 25–48 h PMI groups, which indicated that cTnI, cTnT, and NT-proBNP remained relatively stable under the condition of PMI <48 h.[14] In addition, cryopreservation is a conventional method to prolong the preservation time of samples, and cryopreservation of cadavers can delay the process of decomposition and postmortem changes.[21] Good stability of NT-proBNP, cTnT, and CK-MB under in vitro freezing conditions was demonstrated by several clinical and forensic studies, revealing plasma NT-proBNP can be stably stored at – 20°C for 1 year, and serum cTnT and CK-MB can be stably stored at 4°C for 14 days.[16],[22],[23] According to the results of the above studies, we believe that the levels of NT-proBNP, cTnT and CK-MB in the pericardial fluid samples of either nonfrozen cadavers with PMI <48 h or cadavers with PMI <12 h and freezing time <7 day are relatively stable, and this was taken as one of the inclusion conditions of the present study.

The present study evaluated the levels of NT-proBNP, cTnT, and CK-MB in pericardial fluid of the SCD group and the control group, which revealed that the levels of NT-proBNP, cTnT and CK-MB in pericardial fluid of the SCD group were significantly higher than those in the control group. ROC analysis showed that NT-proBNP, cTnT, and CK-MB in pericardial fluid were with auxiliary diagnostic significance, and the cutoff values for NT-proBNP, cTnT, and CK-MB were 2236 pg/ml, 199.51 ng/ml, and 2742.5 ng/ml, respectively. We also found that the diagnostic efficiency of combined analyses of these three biomarkers was better than each single indicator alone. Therefore, in forensic practice, combined biochemical analyses of NT-proBNP, cTnT, and CK-MB were recommended for those suspected SCD cases to provide more effective information for the postmortem diagnosis.

Further analysis of the levels of NT-proBNP, cTnT, and CK-MB in pericardial fluid among AIHD, AMI, and RMI subgroups as well as between each subgroup and control group showed that the levels of NT-proBNP in the AMI and RMI subgroup were significantly higher than that in the AIHD subgroup and the control group. Zhu et al. reported that BNP levels in the pericardial fluid of the individuals with acute myocardial ischemia did not increase, especially in those with an interval of <0.5 h between the onset of the symptom and death.[15] Michaud et al. also reported that NT-proBNP is useless for diagnosing the early stage of myocardial ischemia.[16] According to the previous studies, levels of NT-proBNP in the AIHD patients would not increase systematically due to the rapid death process and the short survival time. Therefore, levels of NT-proBNP in AIHD subgroup of the present study did not reveal statistical elevation. The levels of cTnT and CK-MB in pericardial fluid in the AIHD, AMI and RMI subgroups were significantly higher than those in control group. Furthermore, the levels of CK-MB in pericardial fluid in the AIHD and RMI subgroups were higher than that in the AMI subgroup. Previous studies demonstrated that myocardium was damaged by reversible ischemia inducing the release of CK-MB at the early stage of myocardial ischemia, and suffered from irreversible ischemia inducing the increasing levels of troponin when the ischemia continued.[24],[25] We speculated that AIHD patients had a short survival time and a rapid death process. Due to the history of old myocardial infarction, the death process of patients with RMI was shorter than that of patients with AMI. Therefore, the pathological changes of myocardium in AIHD and RMI patients were mainly the early-stage myocardial ischemia, accompanied by significant increase of CK-MB level.

Zhu et al. reported that the cTnT/CK-MB ratio in the pericardial fluid and serum from the heart elevated in AMI patients; the ratio was not affected by the prolonging of PMI when PMI was <48 h; the ratio could be an indication for evaluating myocardial damage.[19] In the present study, the cTnT/CK-MB ratio in pericardial fluid showed that the ratio in the AMI subgroup was significantly higher than the AIHD, RMI subgroup and the control group. ROC analysis showed that cTnT/CK-MB ratio might be useful to distinguish AMI from AIHD and RMI at a cutoff value of 0.1085. Therefore, based on combined analysis of NT-proBNP, cTnT, and CK-MB in pericardial fluid, the analysis of the cTnT/CK-MB ratio could further assist diagnosing SCD in forensic practice.

In addition, the cause of death of the individuals in the control group included in the present study was consistent, which was immediate death from traffic accidents, and according to previous reports, the diseases, physical, and chemical factors affecting the pathophysiological process before death were excluded.[15] Furthermore, it is reported that NT-proBNP was found elevated in kidney disease, pulmonary hypertension, pulmonary embolism, and other renal and pulmonary diseases;[26],[27] cTnT was found elevated in causes of death such as hyperthermia, methamphetamine (MA) abuse, carbon monoxide (CO) poisoning, electrocution, psychotropic drug poisoning, pulmonary embolism, late-stage of renal failure, and cerebrovascular diseases;[17],[25],[28],[29] CK-MB was found elevated in asphyxiation, hyperthermia, cerebrovascular disease, pneumonia, electrocution, CO poisoning, fetal MA intoxication, and antipsychotic poisoning.[18],[19],[28],[29] Forensic pathologists should pay attention to the results of the comprehensive analysis mentioned above in practice. It is worth noting that SCD could not be excluded with the low level of NT-proBNP, cTnT, CK-MB, and the cTnT/CK-MB ratio. Therefore, cause of death couldn't be concluded only depend on the result of biochemistry analysis alone for the diagnosis but should be comprehensively analyzed and judged based on the result of the case history, autopsy findings, histological, toxicological, and even imaging methods.

The present study has some limitations. Compared with the SCD group, the number of cases included in the control group was relatively limited, which may limit the accuracy of our research. Moreover, specimens used in the present study were obtained just from one forensic investigation center. The detection data based on the large sample size collected from the multi-center will be very meaningful for the determination of the forensic postmortem reference value of these biomarkers in the future. Moreover, the present retrospective study preliminarily demonstrated these biomarker could assist to diagnose SCD, but further studies of its value for diagnosis of SCD without typical morphological changes are still needed.

 Conclusion



In conclusion, postmortem biochemical analyses of NT-proBNP, cTnT and CK-MB in the pericardial fluid may assist to diagnose SCD in forensic practice, and the combined analyses of multiple biomarkers have better diagnostic efficiency than each single biomarker alone. On the basis of the postmortem, biochemical analyses of NT-proBNP, cTnT and CK-MB, combining the ratio of cTnT/CK-MB could be used to distinguish AMI.

Financial support and sponsorship

This research was funded by the National Natural Science Foundation of China (Grant No. 82002001).

Conflicts of interest

There are no conflicts of interest.

References

1Kandala J, Oommen C, Kern KB. Sudden cardiac death. Br Med Bull 2017;122:5-15.
2Kuriachan VP, Sumner GL, Mitchell LB. Sudden cardiac death. Curr Probl Cardiol 2015;40:133-200.
3Wong CX, Brown A, Lau DH, Chugh SS, Albert CM, Kalman JM, et al. Epidemiology of sudden cardiac death: Global and regional perspectives. Heart Lung Circ 2019;28:6-14.
4Cao Z, Zhao M, Xu C, Zhang T, Jia Y, Wang T, et al. Evaluation of agonal cardiac function for sudden cardiac death in forensic medicine with postmortem brain natriuretic peptide (BNP) and NT-proBNP: A meta-analysis. J Forensic Sci 2020;65:686-91.
5Lawler W. The negative coroner's necropsy: A personal approach and consideration of difficulties. J Clin Pathol 1990;43:977-80.
6Mendis S, Thygesen K, Kuulasmaa K, Giampaoli S, Mähönen M, Ngu Blackett K, et al. World Health Organization definition of myocardial infarction: 2008-09 revision. Int J Epidemiol 2011;40:139-46.
7Shah H, Haridas N. A serial follow up study of cardiac marker enzymes during the week after acute myocardial infarction. Indian J Clin Biochem 2007;22:33-6.
8Maries L, Manitiu I. Diagnostic and prognostic values of B-type natriuretic peptides (BNP) and N-terminal fragment brain natriuretic peptides (NT-pro-BNP). Cardiovasc J Afr 2013;24:286-9.
9Nguyen TL, Phan JA, Hee L, Moses DA, Otton J, Terreblanche OD, et al. High-sensitivity troponin T predicts infarct scar characteristics and adverse left ventricular function by cardiac magnetic resonance imaging early after reperfused acute myocardial infarction. Am Heart J 2015;170:715-25.e2.
10Madea B, Musshoff F. Postmortem biochemistry. Forensic Sci Int 2007;165:165-71.
11Maeda H, Ishikawa T, Michiue T. Forensic biochemistry for functional investigation of death: Concept and practical application. Leg Med (Tokyo) 2011;13:55-67.
12Osuna E, Pérez-Cárceles MD, Alvarez MV, Noguera J, Luna A. Cardiac troponin I (cTn I) and the postmortem diagnosis of myocardial infarction. Int J Legal Med 1998;111:173-6.
13Beausire T, Faouzi M, Palmiere C, Fracasso T, Michaud K. High-sensitive cardiac troponin hs-TnT levels in sudden deaths related to atherosclerotic coronary artery disease. Forensic Sci Int 2018;289:238-43.
14Palmiere C, Tettamanti C, Bonsignore A, De Stefano F, Vanhaebost J, Rousseau G, et al. Cardiac troponins and NT-proBNP in the forensic setting: Overview of sampling site, postmortem interval, cardiopulmonary resuscitation, and review of the literature. Forensic Sci Int 2018;282:211-8.
15Zhu BL, Ishikawa T, Michiue T, Li DR, Zhao D, Tanaka S, et al. Postmortem pericardial natriuretic peptides as markers of cardiac function in medico-legal autopsies. Int J Legal Med 2007;121:28-35.
16Michaud K, Augsburger M, Donzé N, Sabatasso S, Faouzi M, Bollmann M, et al. Evaluation of postmortem measurement of NT-proBNP as a marker for cardiac function. Int J Legal Med 2008;122:415-20.
17Cao Z, Zhao M, Xu C, Zhang T, Jia Y, Wang T, et al. Diagnostic roles of postmortem cTn I and cTn T in cardiac death with special regard to myocardial infarction: A systematic literature review and meta-analysis. Int J Mol Sci 2019;20:3351. [Doi: 10.3390/ijms20133351].
18Xu C, Zhang T, Zhu B, Cao Z. Diagnostic role of postmortem CK-MB in cardiac death: A systematic review and meta-analysis. Forensic Sci Med Pathol 2020;16:287-94.
19Zhu BL, Ishikawa T, Michiue T, Li DR, Zhao D, Bessho Y, et al. 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 (Tokyo) 2007;9:241-50.
20Zhu BL, Ishikawa T, Michiue T, Li DR, Zhao D, Kamikodai Y, et al. 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 (Tokyo) 2006;8:94-101.
21Jia YQ, Jin GD, Tian MH, Xiao Y, Xue JJ, Wang TQ, et al. Effect of corpse cryopreservation on forensic pathological identification. Fa Yi Xue Za Zhi 2019;35:74-7.
22Nowatzke WL, Cole TG. Stability of N-terminal pro-brain natriuretic peptide after storage frozen for one year and after multiple freeze-thaw cycles. Clin Chem 2003;49:1560-2.
23Woltersdorf WW, Bayly GR, Day AP. Practical implications of in vitro stability of cardiac markers. Ann Clin Biochem 2001;38:61-3.
24Gerhardt W, Ljungdahl L, Herbert AK. Troponin-T and CK MB (mass) in early diagnosis of ischemic myocardial injury. The Helsingborg study, 1992. Clin Biochem 1993;26:231-40.
25Zhu BL, Ishikawa T, Michiue T, Li DR, Zhao D, Oritani S, et al. Postmortem cardiac troponin T levels in the blood and pericardial fluid. Part 1. Analysis with special regard to traumatic causes of death. Leg Med (Tokyo) 2006;8:86-93.
26Sabatasso S, Vaucher P, Augsburger M, Donzé N, Mangin P, Michaud K. Sensitivity and specificity of NT-proBNP to detect heart failure at post mortem examination. Int J Legal Med 2011;125:849-56.
27de Lemos JA, McGuire DK, Drazner MH. B-type natriuretic peptide in cardiovascular disease. Lancet 2003;362:316-22.
28Wang Q, Michiue T, Ishikawa T, Zhu BL, Maeda H. 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 (Tokyo) 2011;13:226-32.
29Chen JH, Inamori-Kawamoto O, Michiue T, Ikeda S, Ishikawa T, Maeda H. Cardiac biomarkers in blood, and pericardial and cerebrospinal fluids of forensic autopsy cases: A reassessment with special regard to postmortem interval. Leg Med (Tokyo) 2015;17:343-50.