- Email: [email protected]
Significance of postmortem biochemistry in determining the cause of death
Legal Medicine 11 (2009) S46–S49
Contents lists available at ScienceDirect
Legal Medicine journal homepage: www.elsevier.com/locate/legalmed
Review Article
Significance of postmortem biochemistry in determining the cause of death Hitoshi Maeda a,*, Bao-Li Zhu a,b, Takaki Ishikawa a, Li Quan a, Tomomi Michiue a a b
Department of Legal Medicine, Osaka City University Medical School, Asahi-machi 1-4-3, Abeno, 545-8585 Osaka, Japan Department of Forensic Pathology, China Medical University School of Forensic Medicine, No. 92, Beier Road, Heping District, Shenyang 110001, Liaoning Province, PR China
a r t i c l e
i n f o
Article history: Received 18 December 2008 Accepted 8 January 2009 Available online 6 March 2009 Keywords: Forensic pathology Postmortem blood biochemistry Cause of death Death process Predisposition
a b s t r a c t There have been an abundance of challenging publications on biochemical procedures for investigating death. However, such procedures do not appear to have been effectively incorporated in routine casework. Biochemical profiles at autopsy may show considerable case variations due to various factors involving preexisting disorders, the cause of death, complications, the survival period, and postmortem changes, distributions and localizations of analytes. Postmortem interference may also be caused by various factors, including the status at the time of death, possible supravital reactions, leakage from cell deterioration, diffusion/redistribution, and analytical procedures. Thus, analyses of topographic distribution are also important. When these factors are taken into consideration, biochemical procedures provide useful findings for investigating the cause and process of death, contributory conditions, and predisposing disorders. Meanwhile, recent studies showed that postmortem molecular biological analyses of mRNA of biological reactants in the tissues using RT-PCR are potentially useful for investigating the pathophysiology of death. As above, the use of postmortem biochemistry and molecular biology has advantages for investigating systemic pathophysiological functional changes involved in the dying process. For this purpose, the usefulness of comprehensive analyses of pathological and biochemical findings is suggested as part of laboratory investigations, comprising morphology, toxicology, microbiology, biochemistry and molecular biology, along with diagnostic imaging procedures. These procedures can be effectively incorporated into a ‘full autopsy’ in the context of risk management. The application of these procedures may depend on the concept of medicolegal autopsy, and it is essential to establish postmortem databases through routine casework. Ó 2009 Elsevier Ireland Ltd. All rights reserved.
1. General aspects The essential social and academic tasks of legal medicine include forensic pathological investigations, which involve medicolegal issues including the cause and process of death, especially in cases of traumatic and unexpected sudden death. To meet the social requirements through reliable interpretation of these issues, systematic practical investigations for postmortem assessment of the causes of death and the fatal process using a wide spectrum of markers involving morphology, toxicology, microbiology, biochemistry and molecular biology are necessary. Among these, postmortem biochemistry has become an important ancillary procedure in determining the cause and time of death [1–14]. The possible usefulness of molecular biological investigation has also been described [15–19]. There have been an abundance of challenging publications on biochemical procedures for investigating death. The early attempts used application of clinical markers, followed by the development * Corresponding author. Tel.: +81 6 6645 3767; fax: +81 6 6634 3871. E-mail address: [email protected] (H. Maeda). 1344-6223/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.legalmed.2009.01.048
of new technology mainly for identifying tissue-specific markers. However, such procedures do not appear to have been effectively incorporated in routine casework, partly due to hesitation considering possible interference during the agony and postmortem periods or a classical concept of autopsy (priority of morphology), and also due to a lack of established data. It should also be noted that non-systematic sporadic studies from incidental ideas involving new technologies, which may be very academic, but do not provide practical data, have also hindered further practical challenges. To establish postmortem biochemical and molecular biological data available for medicolegal casework, multicentric serial studies using easily accessible standardized procedures are necessary. The use of these procedures has advantages of standardization, quality assurance, quantitative analyses, statistic assessment and availability of multiple markers, while there are problems involved in the selection and collection of materials and applicability of analytical procedures. The utilization of these procedures is limited to cases in the early postmortem period. It should also be noted that clinical criteria may not be valid in postmortem investigation, not only due to agonal and postmortem interference, but also due to differences in the cause and course of death; medicolegal
H. Maeda et al. / Legal Medicine 11 (2009) S46–S49
investigations are largely involved in cases of preclinical death or cardiopulmonary arrest following traumas and unexpected disease attacks. 2. Properties of postmortem biochemical markers 2.1. Requisitions of postmortem biochemical markers The main purpose of using postmortem biochemistry and molecular biology is to investigate the systemic pathophysiological changes involved in the dying process that cannot usually be detected by morphological methods; these may be called pathophysiological vital reactions. Candidates for these may be practically classified: (a) reactive or degenerative products in the blood and body fluids, (b) immunohistochemical markers in tissues, and (c) molecular biological markers involved in up/down regulations due to the fatal deterioration of homeostasis. Requirements for these markers are: quick responders, postmortem stability, discrimination power (specificity and sensitivity), low cost and easy access, easy and quick analysis for screening, standardization and quality assurance. 2.2. Characteristics of postmortem biochemical profiles Biochemical profiles at autopsy may show considerable case variations due to various factors involving preexisting disorders, the cause of death, complications, the survival period, and postmortem changes that depend on environmental factors and also on the chemical properties, distributions and localizations of analytes (Fig. 1). Postmortem interference with biochemical markers may also be caused by various factors including the status at the time of death, possible supravital reactions, leakage from cell deterioration, diffusion/redistribution dependent on concentration gradients, and analytical procedures. Due to these uncontrollable factors, conventional concepts in postmortem biochemistry appear to be mainly limited to the use of relatively stable markers in the peripheral blood and some body fluids by the application of clinical reference intervals, excluding agonal or postmortem alterations. However, several studies have suggested that positive evaluation may be possible with the use of multiple markers and the setting of postmortem reference intervals, considering the agonal and postmortem changes [1,2]. Analyses of topographic distributions in cardiac and peripheral blood, as well as body fluids, may also be useful [4]. For practical application, it is essential to establish postmortem databases in biochemistry, immunohistochemistry and molecular biology.
Biochemical profile at autopsy Case-to-case variations due to multiple factors
Preexisting disorders
Modification
Cause of death Complications Survival time Status at the time of death
Postmortem changes Environments
Chemical property/Distribution/Localization Fig. 1. Factors contributing to postmortem biochemical profiles.
S47
Table 1 Stability of serum biochemical and molecular biological markers. Examples Stable: urea nitrogen, serum proteins, cholesterol, bilirubin, cholinesterase, cGTP, CRP, erythropoietin Elevation during survival period: creatinine, uric acid, amylase, pulmonary surfactants, catecholamines, S100, neopterin Postmortem elevation: magnesium, myocardial markers Postmortem decrease: sodium, chloride Postmortem elevation and subsequent decrease: calcium
2.3. Classification of biochemical and molecular biological markers in terms of agonal and postmortem interference Comprehensive reviews are available for biochemical markers [1–3], and our serial studies confirmed previous findings for postmortem interference (Table 1): biochemical markers are classified: group 1, stable and comparable to clinical criteria; group 2, predictable agonal/postmortem interference; group 3, unpredictable agonal/postmortem interference [4]. However, it has been suggested that further analyses with regard to the cause of death provide more useful information during postmortem investigation of death. In this respect, postmortem biochemical and molecular biological findings may depend not only on the cause of death, but also on the dying process (Fig. 1). 3. Practical application 3.1. Purposes of postmortem biochemistry Biochemical procedures have been often applied to investigating fatalities without definite pathological evidence, including hypothermia, hyperthermia, electrocution, asphyxiation, drowning, uremia, and acute cardiac death. However, the procedures are also useful for investigating the death process to support and reinforce morphology/toxicology, and for screening morphologically unexpected causes of death in routine casework. 3.2. Conventional markers When the abovementioned factors are taken into consideration, biochemical procedures provide useful findings for investigating the cause and process of death, contributory conditions, and predisposing disorders. For example, postmortem stabilities of urea nitrogen and creatinine in the blood and various body fluids have been established [1–4,7,8]. Their levels in the blood and pericardial fluid usually show an equivalency, and postmortem findings for azotemia (e.g. renal failure and dehydration) can be assessed by the application of clinical criteria [4,7,8]. For drowning cases, however, mildly lower levels in the left heart blood than those in the right suggest the influence of water aspiration. Significant changes in left heart blood levels were also observed for serum sodium, chloride, calcium and magnesium (<48 h postmortem). Furthermore, these changes were well correlated with total lung weight (the sum of combined lung weight and amounts of pleural effusion), thus suggesting the usefulness of combined analyses of pathological and biochemical findings [4]. For uric acid, the pericardial level usually remained within the clinical reference interval, and there were significant elevations in cases of fatal hypothermia and methamphetamine abuse, suggesting prolonged death associated with metabolic deterioration. The right heart blood level was especially elevated in cases of fatal hyperthermia and methamphetamine abuse, which may cause advanced skeletal muscle damage, and also in hypoxic deaths from asphyxiation and drowning [7,8]. Serum creatinine may also be elevated by massive skeletal muscle damage in a short survival period, while the pericardial level remains relatively stable.
S48
H. Maeda et al. / Legal Medicine 11 (2009) S46–S49
Serum proteins are stable, but gradually decrease after massive bleeding and/or tissue damage, while an increase is seen in mechanical asphyxiation. Pulmonary surfactants and S100B protein are tissue-specific serum markers, which are increased immediately after lung and brain damage, respectively, showing a substantial topographic difference. Thus, biochemical findings are useful for death investigation when they are assessed with relevance to pathological findings following thorough autopsy. In terms of recent trends involving the application of biochemical procedures in forensic pathology, there has been great interest in the use of myocardial markers for the postmortem investigation of ischemic heart disease, which is the most common cause of sudden death in many countries and is often very difficult to determine morphologically [20–23]. However, a comprehensive study involving a spectrum of traumatic deaths showed that elevations in postmortem serum and pericardial levels of cardiac troponins may depend on the severity of myocardial damage from various causes of death at the time of death, including hyperthermia, methamphetamine abuse and carbon monoxide poisoning. There were significant relationships to the myocardial pathology and time since death, and there was also a marked topographic difference which depended on the survival time. Thus, cardiac troponin levels should be discussed based on careful comparison with the pathological findings and in considering the postmortem period. Very careful consideration is necessary for the practical validity of an easy ‘point of care’ procedure in postmortem investigations [23].
gradual increase or decrease in the mRNA measurements for target genes. Our studies showed that relative mRNA levels for pulmonary surfactant-associated protein A (SP-A), vascular endothelial growth factor (VEGF), and hypoxia-inducible factor 1A (HIF1A) were stable compared with the reference gene of glyceraldehydes-3-phosphate dehydrogenase (GAPDH) for days after death, while erythropoietin (EPO) mRNA appeared more stable, showing a gradual increase in the measured value. VEGF mRNA was especially stable, and it may be potentially useful for investigating the pathophysiology of death involving acute circulatory deterioration. In addition, the possible application of whole body NMR analyses is suggested.
3.3. Multifaceted markers
4. Significance of biochemistry in routine casework
Several markers can be detected chemically in blood and body fluids or immunohistochemically and/or molecular biologically in tissues. The combined analyses of such multifaceted markers by different procedures may be useful in order to investigate the varied phases of biochemical changes during the dying process; reactive gene expression, de novo synthesis, degradation and leakage from injured cells into the blood stream or body fluids. For example, pulmonary surfactant-associated protein A (SP-A) can be detected chemically in blood, and immunohistochemically and molecular biologically in lung tissues [6,15,24,25]. The most significant findings were immunostaining patterns in the lung tissues, which were useful in investigations of alveolar injury, acute respiratory distress/asphyxia and protracted respiratory failure [24]. The blood biochemistry and molecular biology of SP-A were helpful in supporting the findings of alveolar injury and acute respiratory distress/asphyxia, respectively. In another example, S100 protein is detected chemically in blood and body fluids, and immunohistochemically in brain tissues. Immunopositivity in brain astrocytes was significantly lower in acute deaths due to strangulation/hanging and drowning, whereas the serum level was markedly elevated in cases of strangulation/hanging and acute head injury deaths. A combination of these methods may be useful in investigating the severity and cause of brain damage.
As above, the use of postmortem biochemistry and molecular biology has advantages for investigating systemic pathophysiological functional changes involved in the dying process. For this purpose, the usefulness of comprehensive analyses of pathological and biochemical findings is suggested as part of the laboratory investigations, comprising morphology, toxicology, microbiology, biochemistry and molecular biology, along with diagnostic imaging procedures. These procedures are essential for the pathognomonic assessment of both the cause and process of death (Fig. 2).
3.5. Comprehensive data analyses for investigating the cause and pathophysiology of death Comprehensive analyses of postmortem biochemical and molecular biological findings demonstrated the complex pathophysiology of death, e.g. for asphyxiation: respiratory distress, hypoxia, circulatory disturbance and neurogenic stress responses; for drowning: metabolic and circulatory failure, pulmonary alveolar injury and hypoxia; for fire death: thermal muscle injury, hemolysis, hypoxia, carbon monoxide/other toxic gas poisoning, pulmonary alveolar injury, respiratory and circulatory failure; for hyperthermia: multiple organ failure involving skeletal muscle damage; for sudden cardiac death: myocardial ischemia/necrosis and respiratory distress.
Strategy A. Non-invasive procedures - incl. Diagnostic imaging B. Autopsy and laboratory investigations pathology: macro - and microscopy toxicology microbiology pathophysiochemistry C. Pathophysiogical analyses of the dying process circulatory failure/collapse
3.4. Further approaches Recent advances in molecular biology suggest the potential usefulness of mRNA analyses in the postmortem investigation of fatal mechanisms, although there appears to be a discouraging preconception that such methods are invalid for autopsy materials. For the relative assaying of mRNA transcripts, that of the target gene is usually normalized to that of a house-keeping gene used as an endogenous reference, both of which gradually degrade and decreased with time after death [16]. The difference in postmortem stability between the target and reference genes can cause a
respiratory failure/asphyxiation brain dysfunction inflammatory reaction
D. Pathognomonic assessment E. Medicolegal assessment - incl. clinical/circumstantial evidence Fig. 2. Dynamic assessment of the cause of death.
H. Maeda et al. / Legal Medicine 11 (2009) S46–S49
5. A prospective view In medicolegal practice, biochemistry and molecular biology provide useful support for pathological evidence by ‘visualizing’ pathophysiological alterations and can be effectively incorporated into routine forensic casework in the context of risk management as a part of laboratory investigations involved in a ‘full autopsy service’; the application of these procedures may depend on the concept of medicolegal autopsy. For this purpose, it is essential to establish postmortem databases using standardized procedures through routine casework. Well-coordinated systemic applications of laboratory procedures in collaboration with pathology and diagnostic imaging will be useful for evidence-based assessment in routine casework to meet social requirements, as well as to be available following personal requests through the reliable interpretation of medicolegal issues, including the cause and process of death. However, unpredictable postmortem interference may occur even in the early postmortem period. Nonspecific terminal metabolic deterioration should also be considered. Some serum analytes were markedly elevated compared with clinical levels. One procedure to control these phenomena is multisite investigation of multiple markers. There are various criticisms of the practical use of biochemical and molecular biological procedures. However, application of these procedures may depend on the concept of medicolegal autopsy and systematic peer review. 6. Conclusion The use of postmortem biochemistry and molecular biology has advantages for investigating systemic pathophysiological functional changes involved in the dying process. For this purpose, the usefulness of the comprehensive analyses of pathological and biochemical findings is suggested as a part of laboratory investigations involved in a ‘full autopsy’ in the context of risk management. The application of these procedures may depend on the concept of medicolegal autopsy, and it is essential to establish postmortem databases through routine casework. Conflict of interest The authors have no conflict of interest. Acknowledgements The authors are deeply grateful to coworkers at their institute. This study was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science and the Ministry of Education, Culture, Sports, Science and Technology, Japan (Grant Nos. 11670425 and 12470109, 08307006, 15390217 and 15590585). References [1] Coe JI. Postmortem chemistry of blood, cerebrospinal fluid and vitreous humor. In: Tedeschi CG, Eckert WC, Tedeschi LG, editors. Forensic medicine, vol. 2. Philadelphia: WB Saunders; 1977. p. 1030–60.
S49
[2] Coe JI. Postmortem chemistry update, emphasis on forensic application. Am J Forensic Med Pathol 1993;14:91–117. [3] Friedrich G. Forensische postmortale Biochemie. In: Forster B, editor. Praxis der Rechtsmedizin fuer Mediziner und Juristen. Stuttgart: G Thieme; 1986. p. 789–831. [4] Maeda H. Pathophysiochemistry of acute death: an approach to evidencebased assessment in forensic pathology. Jpn J Legal Med 2004;58:121–9 (in Japanese with English abstract). [5] Zhu B-L, Ishikawa T, Michiue T, Quan L, Maeda H. Postmortem serum endotoxin level in relation to the causes of death. Leg Med 2005;7:103–9. [6] Zhu B-L, Ishida K, Quan L, Li DR, Taniguchi M, Fujita MQ, et al. Pulmonary immunohistochemistry and serum levels of a surfactant-associated protein A in fatal drowning. Leg Med 2002;4:1–6. [7] Zhu B-L, Ishida K, Quan L, Taniguchi M, Oritani S, Li D-R, et al. Postmortem serum uric acid and creatinine levels in relation to the causes of death. Forensic Sci Int 2002;125:59–66. [8] Zhu B-L, Ishikawa T, Michiue T, Li D-R, Zhao D, Quan L, et al. Evaluation of postmortem urea nitrogen, creatinine and uric acid levels in pericardial fluid in forensic autopsy. Leg Med 2005;7:287–92. [9] Li D-R, Zhu B-L, Ishikawa T, Zhao D, Michiue T, Maeda H. Postmortem serum protein S100B levels with regard to the cause of death involving brain damage in medicolegal autopsy cases. Leg Med 2006;8:71–7. [10] Li DR, Zhu B-L, Ishikawa T, Zhao D, Michiue T, Maeda H. Immunohistochemical distribution of S-100 protein in the cerebral cortex with regard to the cause of death in forensic autopsy. Leg Med 2006;8:78–85. [11] Zhu B-L, Ishikawa T, Michiue T, Li D-R, 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 2006;8:86–93. [12] Zhu B-L, Ishikawa T, Michiue T, Li D-R, 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 2006;8:94–101. [13] Zhu B-L, Ishikawa T, Michiue T, Li DR, Zhao D, Quan L, et al. Postmortem serum catecholamine levels in relation to the cause of death. Forensic Sci Int 2007;173:122–9. [14] Zhu B-L, 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. [15] Ishida K, Zhu B-L, Maeda H. A quantitative RT-PCR assay of surfactantassociated protein A1 and A2 mRNA transcripts as a diagnostic tool for acute asphyxial death. Leg Med 2002;4:7–12. [16] Ishida K, Zhu B-L, Maeda H. TaqMan fluorogenic detection system to analyze gene transcription in autopsy materials. In: Keohavong P, Grant SG, editors. Methods in molecular biology, vol. 291. Molecular toxicology protocols. Totowa, NJ: Humana Press; 2004. p. 415–21. [17] Zhao D, Zhu B-L, Ishikawa T, Quan L, Li D-R, Maeda H. Real-time RT-PCR quantitative assays and postmortem degradation profiles of erythropoietin, vascular endothelial growth factor and hypoxia-inducible factor 1 alpha mRNA transcripts in forensic autopsy materials. Leg Med 2006;8:132–6. [18] Zhao D, Zhu B-L, Ishikawa T, Li D-R, Michiue T, Maeda H. Quantitative RT-PCR assays of hypoxia-inducible factor 1a, erythropoietin and vascular endothelial growth factor mRNA transcripts in the kidneys with regard to the cause of death in medicolegal autopsy. Leg Med 2006;8:258–63. [19] Zhu B-L, Tanaka S, Ishikawa T, Zhao D, Li DR, Michiue T, et al. Forensic pathological investigation of myocardial hypoxia-inducible factor-1 alpha, erythropoietin and vascular endothelial growth factor in cardiac death. Leg Med 2008;10:11–9. [20] Dressler J, Felscher D, Koch R, Muller E. Troponin T in legal medicine. Lancet 1998;352:38. [21] Cina SJ, Brown DK, Smialek JE, Collins KA. A rapid postmortem cardiac troponin T assay: laboratory evidence of sudden cardiac death. Am J Forensic Med Pathol 2001;22:173–6. [22] Perez-Carceles MD, Noguera J, Jimenez JL, Martinez P, Luna A, Osuna E. Diagnostic efficacy of biochemical markers in diagnosis postmortem of ischemic heart disease. Forensic Sci Int 2004;142:1–7. [23] Ellingsen CL, Hetland O. Serum concentrations of cardiac troponin T in sudden death. Am J Forensic Med Pathol 2004;25:213–5. [24] Zhu B-L, Ishida K, Fujita MQ, Maeda H. Immunohistochemical investigation of a pulmonary surfactant in fatal mechanical asphyxia. Int J Legal Med 2000;113:268–71. [25] Maeda H, Fujita MQ, Zhu B-L, Ishida K, Quan L, Oritani S, et al. Pulmonary surfactant-associated protein A as a marker of respiratory distress in forensic pathology: assessment of the immunohistochemical and biochemical findings. Leg Med 2003;5:S318–21.
Contents lists available at ScienceDirect
Legal Medicine journal homepage: www.elsevier.com/locate/legalmed
Review Article
Significance of postmortem biochemistry in determining the cause of death Hitoshi Maeda a,*, Bao-Li Zhu a,b, Takaki Ishikawa a, Li Quan a, Tomomi Michiue a a b
Department of Legal Medicine, Osaka City University Medical School, Asahi-machi 1-4-3, Abeno, 545-8585 Osaka, Japan Department of Forensic Pathology, China Medical University School of Forensic Medicine, No. 92, Beier Road, Heping District, Shenyang 110001, Liaoning Province, PR China
a r t i c l e
i n f o
Article history: Received 18 December 2008 Accepted 8 January 2009 Available online 6 March 2009 Keywords: Forensic pathology Postmortem blood biochemistry Cause of death Death process Predisposition
a b s t r a c t There have been an abundance of challenging publications on biochemical procedures for investigating death. However, such procedures do not appear to have been effectively incorporated in routine casework. Biochemical profiles at autopsy may show considerable case variations due to various factors involving preexisting disorders, the cause of death, complications, the survival period, and postmortem changes, distributions and localizations of analytes. Postmortem interference may also be caused by various factors, including the status at the time of death, possible supravital reactions, leakage from cell deterioration, diffusion/redistribution, and analytical procedures. Thus, analyses of topographic distribution are also important. When these factors are taken into consideration, biochemical procedures provide useful findings for investigating the cause and process of death, contributory conditions, and predisposing disorders. Meanwhile, recent studies showed that postmortem molecular biological analyses of mRNA of biological reactants in the tissues using RT-PCR are potentially useful for investigating the pathophysiology of death. As above, the use of postmortem biochemistry and molecular biology has advantages for investigating systemic pathophysiological functional changes involved in the dying process. For this purpose, the usefulness of comprehensive analyses of pathological and biochemical findings is suggested as part of laboratory investigations, comprising morphology, toxicology, microbiology, biochemistry and molecular biology, along with diagnostic imaging procedures. These procedures can be effectively incorporated into a ‘full autopsy’ in the context of risk management. The application of these procedures may depend on the concept of medicolegal autopsy, and it is essential to establish postmortem databases through routine casework. Ó 2009 Elsevier Ireland Ltd. All rights reserved.
1. General aspects The essential social and academic tasks of legal medicine include forensic pathological investigations, which involve medicolegal issues including the cause and process of death, especially in cases of traumatic and unexpected sudden death. To meet the social requirements through reliable interpretation of these issues, systematic practical investigations for postmortem assessment of the causes of death and the fatal process using a wide spectrum of markers involving morphology, toxicology, microbiology, biochemistry and molecular biology are necessary. Among these, postmortem biochemistry has become an important ancillary procedure in determining the cause and time of death [1–14]. The possible usefulness of molecular biological investigation has also been described [15–19]. There have been an abundance of challenging publications on biochemical procedures for investigating death. The early attempts used application of clinical markers, followed by the development * Corresponding author. Tel.: +81 6 6645 3767; fax: +81 6 6634 3871. E-mail address: [email protected] (H. Maeda). 1344-6223/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.legalmed.2009.01.048
of new technology mainly for identifying tissue-specific markers. However, such procedures do not appear to have been effectively incorporated in routine casework, partly due to hesitation considering possible interference during the agony and postmortem periods or a classical concept of autopsy (priority of morphology), and also due to a lack of established data. It should also be noted that non-systematic sporadic studies from incidental ideas involving new technologies, which may be very academic, but do not provide practical data, have also hindered further practical challenges. To establish postmortem biochemical and molecular biological data available for medicolegal casework, multicentric serial studies using easily accessible standardized procedures are necessary. The use of these procedures has advantages of standardization, quality assurance, quantitative analyses, statistic assessment and availability of multiple markers, while there are problems involved in the selection and collection of materials and applicability of analytical procedures. The utilization of these procedures is limited to cases in the early postmortem period. It should also be noted that clinical criteria may not be valid in postmortem investigation, not only due to agonal and postmortem interference, but also due to differences in the cause and course of death; medicolegal
H. Maeda et al. / Legal Medicine 11 (2009) S46–S49
investigations are largely involved in cases of preclinical death or cardiopulmonary arrest following traumas and unexpected disease attacks. 2. Properties of postmortem biochemical markers 2.1. Requisitions of postmortem biochemical markers The main purpose of using postmortem biochemistry and molecular biology is to investigate the systemic pathophysiological changes involved in the dying process that cannot usually be detected by morphological methods; these may be called pathophysiological vital reactions. Candidates for these may be practically classified: (a) reactive or degenerative products in the blood and body fluids, (b) immunohistochemical markers in tissues, and (c) molecular biological markers involved in up/down regulations due to the fatal deterioration of homeostasis. Requirements for these markers are: quick responders, postmortem stability, discrimination power (specificity and sensitivity), low cost and easy access, easy and quick analysis for screening, standardization and quality assurance. 2.2. Characteristics of postmortem biochemical profiles Biochemical profiles at autopsy may show considerable case variations due to various factors involving preexisting disorders, the cause of death, complications, the survival period, and postmortem changes that depend on environmental factors and also on the chemical properties, distributions and localizations of analytes (Fig. 1). Postmortem interference with biochemical markers may also be caused by various factors including the status at the time of death, possible supravital reactions, leakage from cell deterioration, diffusion/redistribution dependent on concentration gradients, and analytical procedures. Due to these uncontrollable factors, conventional concepts in postmortem biochemistry appear to be mainly limited to the use of relatively stable markers in the peripheral blood and some body fluids by the application of clinical reference intervals, excluding agonal or postmortem alterations. However, several studies have suggested that positive evaluation may be possible with the use of multiple markers and the setting of postmortem reference intervals, considering the agonal and postmortem changes [1,2]. Analyses of topographic distributions in cardiac and peripheral blood, as well as body fluids, may also be useful [4]. For practical application, it is essential to establish postmortem databases in biochemistry, immunohistochemistry and molecular biology.
Biochemical profile at autopsy Case-to-case variations due to multiple factors
Preexisting disorders
Modification
Cause of death Complications Survival time Status at the time of death
Postmortem changes Environments
Chemical property/Distribution/Localization Fig. 1. Factors contributing to postmortem biochemical profiles.
S47
Table 1 Stability of serum biochemical and molecular biological markers. Examples Stable: urea nitrogen, serum proteins, cholesterol, bilirubin, cholinesterase, cGTP, CRP, erythropoietin Elevation during survival period: creatinine, uric acid, amylase, pulmonary surfactants, catecholamines, S100, neopterin Postmortem elevation: magnesium, myocardial markers Postmortem decrease: sodium, chloride Postmortem elevation and subsequent decrease: calcium
2.3. Classification of biochemical and molecular biological markers in terms of agonal and postmortem interference Comprehensive reviews are available for biochemical markers [1–3], and our serial studies confirmed previous findings for postmortem interference (Table 1): biochemical markers are classified: group 1, stable and comparable to clinical criteria; group 2, predictable agonal/postmortem interference; group 3, unpredictable agonal/postmortem interference [4]. However, it has been suggested that further analyses with regard to the cause of death provide more useful information during postmortem investigation of death. In this respect, postmortem biochemical and molecular biological findings may depend not only on the cause of death, but also on the dying process (Fig. 1). 3. Practical application 3.1. Purposes of postmortem biochemistry Biochemical procedures have been often applied to investigating fatalities without definite pathological evidence, including hypothermia, hyperthermia, electrocution, asphyxiation, drowning, uremia, and acute cardiac death. However, the procedures are also useful for investigating the death process to support and reinforce morphology/toxicology, and for screening morphologically unexpected causes of death in routine casework. 3.2. Conventional markers When the abovementioned factors are taken into consideration, biochemical procedures provide useful findings for investigating the cause and process of death, contributory conditions, and predisposing disorders. For example, postmortem stabilities of urea nitrogen and creatinine in the blood and various body fluids have been established [1–4,7,8]. Their levels in the blood and pericardial fluid usually show an equivalency, and postmortem findings for azotemia (e.g. renal failure and dehydration) can be assessed by the application of clinical criteria [4,7,8]. For drowning cases, however, mildly lower levels in the left heart blood than those in the right suggest the influence of water aspiration. Significant changes in left heart blood levels were also observed for serum sodium, chloride, calcium and magnesium (<48 h postmortem). Furthermore, these changes were well correlated with total lung weight (the sum of combined lung weight and amounts of pleural effusion), thus suggesting the usefulness of combined analyses of pathological and biochemical findings [4]. For uric acid, the pericardial level usually remained within the clinical reference interval, and there were significant elevations in cases of fatal hypothermia and methamphetamine abuse, suggesting prolonged death associated with metabolic deterioration. The right heart blood level was especially elevated in cases of fatal hyperthermia and methamphetamine abuse, which may cause advanced skeletal muscle damage, and also in hypoxic deaths from asphyxiation and drowning [7,8]. Serum creatinine may also be elevated by massive skeletal muscle damage in a short survival period, while the pericardial level remains relatively stable.
S48
H. Maeda et al. / Legal Medicine 11 (2009) S46–S49
Serum proteins are stable, but gradually decrease after massive bleeding and/or tissue damage, while an increase is seen in mechanical asphyxiation. Pulmonary surfactants and S100B protein are tissue-specific serum markers, which are increased immediately after lung and brain damage, respectively, showing a substantial topographic difference. Thus, biochemical findings are useful for death investigation when they are assessed with relevance to pathological findings following thorough autopsy. In terms of recent trends involving the application of biochemical procedures in forensic pathology, there has been great interest in the use of myocardial markers for the postmortem investigation of ischemic heart disease, which is the most common cause of sudden death in many countries and is often very difficult to determine morphologically [20–23]. However, a comprehensive study involving a spectrum of traumatic deaths showed that elevations in postmortem serum and pericardial levels of cardiac troponins may depend on the severity of myocardial damage from various causes of death at the time of death, including hyperthermia, methamphetamine abuse and carbon monoxide poisoning. There were significant relationships to the myocardial pathology and time since death, and there was also a marked topographic difference which depended on the survival time. Thus, cardiac troponin levels should be discussed based on careful comparison with the pathological findings and in considering the postmortem period. Very careful consideration is necessary for the practical validity of an easy ‘point of care’ procedure in postmortem investigations [23].
gradual increase or decrease in the mRNA measurements for target genes. Our studies showed that relative mRNA levels for pulmonary surfactant-associated protein A (SP-A), vascular endothelial growth factor (VEGF), and hypoxia-inducible factor 1A (HIF1A) were stable compared with the reference gene of glyceraldehydes-3-phosphate dehydrogenase (GAPDH) for days after death, while erythropoietin (EPO) mRNA appeared more stable, showing a gradual increase in the measured value. VEGF mRNA was especially stable, and it may be potentially useful for investigating the pathophysiology of death involving acute circulatory deterioration. In addition, the possible application of whole body NMR analyses is suggested.
3.3. Multifaceted markers
4. Significance of biochemistry in routine casework
Several markers can be detected chemically in blood and body fluids or immunohistochemically and/or molecular biologically in tissues. The combined analyses of such multifaceted markers by different procedures may be useful in order to investigate the varied phases of biochemical changes during the dying process; reactive gene expression, de novo synthesis, degradation and leakage from injured cells into the blood stream or body fluids. For example, pulmonary surfactant-associated protein A (SP-A) can be detected chemically in blood, and immunohistochemically and molecular biologically in lung tissues [6,15,24,25]. The most significant findings were immunostaining patterns in the lung tissues, which were useful in investigations of alveolar injury, acute respiratory distress/asphyxia and protracted respiratory failure [24]. The blood biochemistry and molecular biology of SP-A were helpful in supporting the findings of alveolar injury and acute respiratory distress/asphyxia, respectively. In another example, S100 protein is detected chemically in blood and body fluids, and immunohistochemically in brain tissues. Immunopositivity in brain astrocytes was significantly lower in acute deaths due to strangulation/hanging and drowning, whereas the serum level was markedly elevated in cases of strangulation/hanging and acute head injury deaths. A combination of these methods may be useful in investigating the severity and cause of brain damage.
As above, the use of postmortem biochemistry and molecular biology has advantages for investigating systemic pathophysiological functional changes involved in the dying process. For this purpose, the usefulness of comprehensive analyses of pathological and biochemical findings is suggested as part of the laboratory investigations, comprising morphology, toxicology, microbiology, biochemistry and molecular biology, along with diagnostic imaging procedures. These procedures are essential for the pathognomonic assessment of both the cause and process of death (Fig. 2).
3.5. Comprehensive data analyses for investigating the cause and pathophysiology of death Comprehensive analyses of postmortem biochemical and molecular biological findings demonstrated the complex pathophysiology of death, e.g. for asphyxiation: respiratory distress, hypoxia, circulatory disturbance and neurogenic stress responses; for drowning: metabolic and circulatory failure, pulmonary alveolar injury and hypoxia; for fire death: thermal muscle injury, hemolysis, hypoxia, carbon monoxide/other toxic gas poisoning, pulmonary alveolar injury, respiratory and circulatory failure; for hyperthermia: multiple organ failure involving skeletal muscle damage; for sudden cardiac death: myocardial ischemia/necrosis and respiratory distress.
Strategy A. Non-invasive procedures - incl. Diagnostic imaging B. Autopsy and laboratory investigations pathology: macro - and microscopy toxicology microbiology pathophysiochemistry C. Pathophysiogical analyses of the dying process circulatory failure/collapse
3.4. Further approaches Recent advances in molecular biology suggest the potential usefulness of mRNA analyses in the postmortem investigation of fatal mechanisms, although there appears to be a discouraging preconception that such methods are invalid for autopsy materials. For the relative assaying of mRNA transcripts, that of the target gene is usually normalized to that of a house-keeping gene used as an endogenous reference, both of which gradually degrade and decreased with time after death [16]. The difference in postmortem stability between the target and reference genes can cause a
respiratory failure/asphyxiation brain dysfunction inflammatory reaction
D. Pathognomonic assessment E. Medicolegal assessment - incl. clinical/circumstantial evidence Fig. 2. Dynamic assessment of the cause of death.
H. Maeda et al. / Legal Medicine 11 (2009) S46–S49
5. A prospective view In medicolegal practice, biochemistry and molecular biology provide useful support for pathological evidence by ‘visualizing’ pathophysiological alterations and can be effectively incorporated into routine forensic casework in the context of risk management as a part of laboratory investigations involved in a ‘full autopsy service’; the application of these procedures may depend on the concept of medicolegal autopsy. For this purpose, it is essential to establish postmortem databases using standardized procedures through routine casework. Well-coordinated systemic applications of laboratory procedures in collaboration with pathology and diagnostic imaging will be useful for evidence-based assessment in routine casework to meet social requirements, as well as to be available following personal requests through the reliable interpretation of medicolegal issues, including the cause and process of death. However, unpredictable postmortem interference may occur even in the early postmortem period. Nonspecific terminal metabolic deterioration should also be considered. Some serum analytes were markedly elevated compared with clinical levels. One procedure to control these phenomena is multisite investigation of multiple markers. There are various criticisms of the practical use of biochemical and molecular biological procedures. However, application of these procedures may depend on the concept of medicolegal autopsy and systematic peer review. 6. Conclusion The use of postmortem biochemistry and molecular biology has advantages for investigating systemic pathophysiological functional changes involved in the dying process. For this purpose, the usefulness of the comprehensive analyses of pathological and biochemical findings is suggested as a part of laboratory investigations involved in a ‘full autopsy’ in the context of risk management. The application of these procedures may depend on the concept of medicolegal autopsy, and it is essential to establish postmortem databases through routine casework. Conflict of interest The authors have no conflict of interest. Acknowledgements The authors are deeply grateful to coworkers at their institute. This study was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science and the Ministry of Education, Culture, Sports, Science and Technology, Japan (Grant Nos. 11670425 and 12470109, 08307006, 15390217 and 15590585). References [1] Coe JI. Postmortem chemistry of blood, cerebrospinal fluid and vitreous humor. In: Tedeschi CG, Eckert WC, Tedeschi LG, editors. Forensic medicine, vol. 2. Philadelphia: WB Saunders; 1977. p. 1030–60.
S49
[2] Coe JI. Postmortem chemistry update, emphasis on forensic application. Am J Forensic Med Pathol 1993;14:91–117. [3] Friedrich G. Forensische postmortale Biochemie. In: Forster B, editor. Praxis der Rechtsmedizin fuer Mediziner und Juristen. Stuttgart: G Thieme; 1986. p. 789–831. [4] Maeda H. Pathophysiochemistry of acute death: an approach to evidencebased assessment in forensic pathology. Jpn J Legal Med 2004;58:121–9 (in Japanese with English abstract). [5] Zhu B-L, Ishikawa T, Michiue T, Quan L, Maeda H. Postmortem serum endotoxin level in relation to the causes of death. Leg Med 2005;7:103–9. [6] Zhu B-L, Ishida K, Quan L, Li DR, Taniguchi M, Fujita MQ, et al. Pulmonary immunohistochemistry and serum levels of a surfactant-associated protein A in fatal drowning. Leg Med 2002;4:1–6. [7] Zhu B-L, Ishida K, Quan L, Taniguchi M, Oritani S, Li D-R, et al. Postmortem serum uric acid and creatinine levels in relation to the causes of death. Forensic Sci Int 2002;125:59–66. [8] Zhu B-L, Ishikawa T, Michiue T, Li D-R, Zhao D, Quan L, et al. Evaluation of postmortem urea nitrogen, creatinine and uric acid levels in pericardial fluid in forensic autopsy. Leg Med 2005;7:287–92. [9] Li D-R, Zhu B-L, Ishikawa T, Zhao D, Michiue T, Maeda H. Postmortem serum protein S100B levels with regard to the cause of death involving brain damage in medicolegal autopsy cases. Leg Med 2006;8:71–7. [10] Li DR, Zhu B-L, Ishikawa T, Zhao D, Michiue T, Maeda H. Immunohistochemical distribution of S-100 protein in the cerebral cortex with regard to the cause of death in forensic autopsy. Leg Med 2006;8:78–85. [11] Zhu B-L, Ishikawa T, Michiue T, Li D-R, 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 2006;8:86–93. [12] Zhu B-L, Ishikawa T, Michiue T, Li D-R, 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 2006;8:94–101. [13] Zhu B-L, Ishikawa T, Michiue T, Li DR, Zhao D, Quan L, et al. Postmortem serum catecholamine levels in relation to the cause of death. Forensic Sci Int 2007;173:122–9. [14] Zhu B-L, 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. [15] Ishida K, Zhu B-L, Maeda H. A quantitative RT-PCR assay of surfactantassociated protein A1 and A2 mRNA transcripts as a diagnostic tool for acute asphyxial death. Leg Med 2002;4:7–12. [16] Ishida K, Zhu B-L, Maeda H. TaqMan fluorogenic detection system to analyze gene transcription in autopsy materials. In: Keohavong P, Grant SG, editors. Methods in molecular biology, vol. 291. Molecular toxicology protocols. Totowa, NJ: Humana Press; 2004. p. 415–21. [17] Zhao D, Zhu B-L, Ishikawa T, Quan L, Li D-R, Maeda H. Real-time RT-PCR quantitative assays and postmortem degradation profiles of erythropoietin, vascular endothelial growth factor and hypoxia-inducible factor 1 alpha mRNA transcripts in forensic autopsy materials. Leg Med 2006;8:132–6. [18] Zhao D, Zhu B-L, Ishikawa T, Li D-R, Michiue T, Maeda H. Quantitative RT-PCR assays of hypoxia-inducible factor 1a, erythropoietin and vascular endothelial growth factor mRNA transcripts in the kidneys with regard to the cause of death in medicolegal autopsy. Leg Med 2006;8:258–63. [19] Zhu B-L, Tanaka S, Ishikawa T, Zhao D, Li DR, Michiue T, et al. Forensic pathological investigation of myocardial hypoxia-inducible factor-1 alpha, erythropoietin and vascular endothelial growth factor in cardiac death. Leg Med 2008;10:11–9. [20] Dressler J, Felscher D, Koch R, Muller E. Troponin T in legal medicine. Lancet 1998;352:38. [21] Cina SJ, Brown DK, Smialek JE, Collins KA. A rapid postmortem cardiac troponin T assay: laboratory evidence of sudden cardiac death. Am J Forensic Med Pathol 2001;22:173–6. [22] Perez-Carceles MD, Noguera J, Jimenez JL, Martinez P, Luna A, Osuna E. Diagnostic efficacy of biochemical markers in diagnosis postmortem of ischemic heart disease. Forensic Sci Int 2004;142:1–7. [23] Ellingsen CL, Hetland O. Serum concentrations of cardiac troponin T in sudden death. Am J Forensic Med Pathol 2004;25:213–5. [24] Zhu B-L, Ishida K, Fujita MQ, Maeda H. Immunohistochemical investigation of a pulmonary surfactant in fatal mechanical asphyxia. Int J Legal Med 2000;113:268–71. [25] Maeda H, Fujita MQ, Zhu B-L, Ishida K, Quan L, Oritani S, et al. Pulmonary surfactant-associated protein A as a marker of respiratory distress in forensic pathology: assessment of the immunohistochemical and biochemical findings. Leg Med 2003;5:S318–21.