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Accurate estimation of the postmortem interval (PMI) has been one of the most important and complicated issues in the forensic practice. In order to provide novel perspectives for the future research concerning PMI, the advantages and disadvantages of related traditional methods, postmortem degradation of nucleic acid and tissue, the componential change of vitreous humor and histological biochemistry since 2002 have been introduced and compared in this review.

Previous studies have suggested the usefulness of the postmortem serum S100B level as a marker of the severity of brain damage. In this study, we investigated the S100B level in the cerebrospinal fluid (CSF) in serial autopsy cases (n=216, within 3 days postmortem), including those of blunt injury (n=34: fatal head injury, n=20; others, n=14), sharp instrument injury (n=9), mechanical asphyxiation (n=19), drowning (n=11), fire fatality (n=26), intoxication (n=20), hypothermia (cold exposure, n=16), hyperthermia (heat stroke, n=9), acute cardiac death (n=52) and pneumonia (n=20). The CSF S100B level showed a moderate postmortem time-dependent increase for acute cardiac death (r=0.58, p<0.0001) and asphyxia (r=0.741, p<0.001). In cases of survival time within 48 h, drowning and hypothermia usually showed a lower CSF S100B level (around 500 ng/ml), and the level was higher for delayed head injury death, asphyxia, intoxication, and hyperthermia (around 1500 ng/ml) (p<0.05). In fatal head injury cases, however, CSF S100B did not correlate with the survival time or postmortem interval. A CSF S100B level of >2000 ng/ml in the early postmortem period might be considered a biochemical sign of fatally severe brain damage.

Previous studies have suggested the usefulness of postmortem serum calcium (Ca) and magnesium (Mg) for investigating cause of death. The present study investigated their levels in the pericardial fluid of serial autopsy cases of adults within 48 h postmortem (n=385), including fatalities from blunt injury (n=57), sharp instrument injury (n=9), mechanical asphyxiation (n=28), salt- and freshwater drowning (n=14 and n=61, respectively), fire fatality (n=35), intoxication (n=23), hypothermia (cold exposure, n=12), hyperthermia (heat stroke, n=7), acute cardiac death (ACD, n=86), pneumonia (n=9) and spontaneous cerebral hemorrhage (n=11). The pericardial Ca level was independent of the postmortem interval, showing a value similar to that of the clinical reference range in cases other than saltwater drowning, while the Mg level was higher than the clinical reference range and showed a mild postmortem time-dependent increase. Pericardial Ca was significantly higher for saltwater drowning than other groups, and a lower level was seen for hyperthermia, and some cases of blunt injury and intoxication. The Mg level was also significantly higher for saltwater drowning than the other groups, and showed a higher level for sharp instrument injury, but a lower level for hypothermia. The Mg/Ca ratio was higher for sharp instrument injury and saltwater drowning, but was lower for hypothermia. These findings suggest that postmortem pericardial Ca and Mg can be used to investigate the cause of death, especially for saltwater drowning, hypothermia and hyperthermia.

The cellular immune response is accompanied by the release of neopterin. The level of neopterin in serum is increased in patients suffering from viral infections, autoimmune diseases, systemic inflammation, allograft rejection and malignant diseases, while that of C-reactive protein (CRP) is known to rise during inflammatory diseases and traumas. To investigate postmortem neopterin and CRP concentrations with regard to the cause of death, we examined cardiac and peripheral blood samples in 474 autopsy cases without advanced decomposition (0-96 years of age, 343 males and 131 females), 2.8 h to 3 days (median, 18.0 h) after death. Survival time was 0.1 h to 5 months (median, 3.0 h) for traumatic death, and 0.1-1, 440 h (median, 2.5 h) for natural death. In autopsied subjects, neopterin concentrations were higher than the clinical reference, independent of the time after death, and depended on the survival time. In cases of acute and subacute death due to trauma, the neopterin level in right heart blood was mildly to moderately elevated (about 50-200 nmol/l) except for sharp instrument injury, whereas the CRP concentration usually remained low (<1 mg/dl). However, a moderate rise in the CRP level (around 1-10 mg/dl) was observed in fatal cases of hypothermia (cold exposure). Markedly elevated serum CRP and neopterin levels (>10 mg/dl and >500 nmol/l, respectively) were detected in cases of delayed death due to trauma involving systemic inflammatory response syndrome (SIRS) and of fatal bacterial infections. For sepsis, the serum CRP level was markedly elevated but the neopterin level was low in some cases. Fatal viral infections usually resulted in a marked elevation in the serum neopterin level (>500 nmol/l) with a mild to moderate rise in the CRP level. Combined analyses of neopterin and CRP may be useful to investigate viral infections and delayed traumatic death involving SIRS to support pathological findings.

The present study analyzed serum levels of urea nitrogen (UN), creatinine (Cr), and C-reactive protein (CRP), which are very stable during the early postmortem period, for investigation of the cause of death with special regard to hyperthermia (heat stroke) in serial medico-legal autopsy cases (n = 429), excluding fatal injury, intoxication, and fire fatality. In this series, mechanical asphyxiation, drowning, and sudden cardiac death cases (n = 56, n = 43, and n = 212, respectively) usually showed low levels within postmortem reference ranges for these serum markers, although UN and CRP levels were mildly elevated in cases of sudden cardiac death and cerebrovascular stroke. There were concomitant significant elevations in serum levels of UN (>50 mg/dL), Cr (>2 mg/dL), and CRP (>2 mg/dL) for chronic renal failure, gastrointestinal bleeding, pneumonia, and hypothermia (cold exposure). UN and CRP were especially high for chronic renal failure and pneumonia, respectively. However, hyperthermia cases showed an isolated elevation in the serum Cr level, suggesting an influence of systemic skeletal muscle damage. These serum markers may be practically useful for postmortem investigation of death due to hyperthermia (heat stroke), for which specific pathological and toxicological evidence may not be available.

Catecholamines are major humoral factors and neurotransmitters that contribute to various stress responses. However, they have been considered unstable due to agony, terminal medical care and postmortem interference. The present study was a comprehensive investigation of postmortem serum levels of adrenaline (Adr), noradrenaline (Nad) and dopamine (DA) with regard to the cause of death in serial medicolegal autopsy cases (n=542) including fatalities from various traumas and diseases. There was a slight tendency toward postmortem increases of Nad and DA in cardiac blood as well as Adr and Nad in peripheral blood, a slight age-dependent decrease in Adr and DA in right heart blood, and a marked increase in serum DA due to administration during critical medical care. When these factors were taken into consideration, significantly higher cardiac blood levels were observed for Adr and Nad in injury and asphyxiation cases and for Adr in fatal methamphetamine (MA) abuse and other poisoning cases, whereas those levels were lower in fatal hypothermia. Drowning, fire fatality, acute cardiac death and cerebrovascular disease showed intermediate Adr and Nad levels. The DA level was elevated in cases of injury, hyperthermia, MA fatality and other poisoning. Topographical analyses suggested that the major sources of increased serum catecholamines in cases of injury was abdominal viscera including adrenal glands, and that in cases of asphyxiation, drowning, fire fatality, hyperthermia, MA fatality, other poisoning, acute cardiac death and cerebrovascular disease was the extremities in addition to abdominal viscera. However, there was in part a large case-to-case difference in each marker related to individual causes of death. These findings differed markedly from clinical observations and suggest that the postmortem serum catecholamine levels may reflect the magnitude of physical stress responses during the process of death in individual cases.

Previous studies showed significant differences in postmortem urea nitrogen (UN), creatinine (Cr) and uric acid (UA) levels in heart blood depending on the causes of death, including acute death. In addition, the levels in pericardial fluid approximated the clinical serum reference ranges, and their elevations may be assessed based on clinical criteria. The present study investigated difference between blood and pericardial levels of these markers. Medicolegal autopsy cases (n=556, within 48h postmortem) of the following causes of death were examined: injury (n=136), asphyxiation (n=50), drowning (n=39), fire fatalities (n=99), hyperthermia (n=11), hypothermia (n=8), poisoning (n=26), delayed traumatic death (n=44) and natural diseases (n=143). When serum UN, Cr and UA levels were compared with the pericardial levels, there was an equivalency for delayed traumatic death and chronic renal failure, although each level was markedly elevated. Parallel increases in serum and pericardial UA and/or Cr levels were also observed for hypothermia and gastrointestinal bleeding. However, in drowning cases, the left cardiac and pericardial UN levels were lower than the right cardiac and peripheral levels, suggesting the influence of water aspiration. Significant elevations in serum and pericardial Cr and UA levels with a higher serum/pericardial UA ratio for fatal methamphetamine intoxication suggest progressive skeletal muscle damage due to advanced hypoxia/acidosis. Similar findings were often observed for other acute and subacute deaths. These findings suggest that a comparison between blood and pericardial nitrogenous compounds would be useful for investigating the cause and process of death.

In forensic pathology, previous studies have suggested the possible application of cardiac troponins in the diagnosis of myocardial infarction. However, there appears to be insufficient practical data on other causes of death. The present study was a comprehensive analysis of the cardiac, peripheral blood and pericardial levels of cardiac troponin T (cTnT) in serial medicolegal autopsy cases (n = 405) with a survival time <24 h and within 48 h postmortem to assess the validity of investigating myocardial damage with special regard to traumatic causes of death. These included blunt and sharp instrument injury (n = 122 and 21, respectively), asphyxiation (n = 35), drowning (n = 27), fire fatalities (n = 94), hyperthermia (n = 13), hypothermia (n = 6), fatal methamphetamine (MA) abuse (n = 12) and carbon monoxide (CO) poisoning (n = 5) in comparison with myocardial infarction (MI, n = 57) and cerebrovascular diseases (n = 13). Cases within 12h postmortem usually showed lower cardiac and pericardial cTnT levels than did those of longer postmortem time of 12-48 h. In the early postmortem period of <12 h, significantly elevated serum cTnT levels were observed for hyperthermia. Thereafter, fatal MA abuse, CO poisoning and MI cases also showed higher levels. However, cTnT remained at lower levels for hypothermia and drowning. The elevation of cTnT was associated with the pathology of advanced myocardial damage involving swelling and liquefactive necrosis. The above-mentioned differences were the smallest for peripheral blood. These findings suggest that elevations in postmortem serum and pericardial cTnT levels depend on the severity of myocardial damage at the time of death and are related to the pathological findings, although postmortem interference should be taken into consideration.

Although previous forensic pathological studies have suggested the possible application of cardiac troponins in the diagnosis of myocardial infarction, there appears to be insufficient data with regard to its cardiac pathology. The present study analyzed the heart blood, peripheral blood and pericardial fluid levels of cardiac troponin T (cTnT) in sudden cardiac deaths (n = 96) within 48h postmortem in relation to pathological findings of acute myocardial infarction (AMI, n = 34), recurrent myocardial infarction (RMI, n = 23), ischemic heart disease without any pathological evidence of infarction (IHD, n = 24) and other heart diseases (OHD, n = 15). Control groups (n = 75, survival time <24 h) within 48 h postmortem consisted of asphyxiation (n = 35), drowning (n = 27) and cerebrovascular diseases (n = 13). There was a marked correlation in the cTnT levels between right and left heart blood samples. The pericardial level was usually higher than either heart blood level, and the external iliac venous blood level was the lowest. Although postmortem time-dependent increases in heart and pericardial blood cTnT levels were observed in most groups, they were most evident for AMI and asphyxiation. In the early postmortem period (<12 h) there was no significant difference between AMI or RMI and the other groups except for drowning. After 12 h postmortem, significantly elevated heart blood and pericardial cTnT levels were observed for AMI and RMI showing multiple interstitial hemorrhages and necrosis compared to those with localized eosinophilic changes or patchy interstitial hemorrhages, IHD and OHD. These differences were the smallest for peripheral blood. For sudden cardiac death cases, the difference in cTnT level at each site among the causes of death was independent of gender, age, heart or lung weight and pathologies of affected coronary artery and severity of coronary stenosis. These observations suggest that the elevation in postmortem blood and pericardial cTnT levels in sudden cardiac death may depend on the severity of ischemic myocardial damage including the size and intensity of myocardial lesions involving multiple interstitial hemorrhages and necrosis, and also the postmortem period for heart and pericardial levels.

Recent advances in molecular biology have suggested the potential usefulness of mRNA analyses in postmortem investigations of fatal mechanisms. The aim of the present study was to establish quantitative assays of oxygen-regulated factors including erythropoietin (EPO), vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1 alpha (HIF1A) mRNAs, and to investigate the postmortem stability of these mRNA transcripts in forensic autopsy materials. Relative quantification of EPO, VEGF and HIF1A mRNAs, based on the TaqMan reverse transcription-polymerase chain reaction (RT-PCR), was performed on autopsy tissue specimens from the heart (n = 10), brain (n = 10), kidney (n = 16) and lung (n = 8) after preservation at room temperature for various storage times. VEGF and HIF1A mRNA gradually degraded in patterns similar to glyceraldehydes-3-phosphate dehydrogenase (GAPDH) mRNA used as an endogenous reference. Accordingly, the relative quantification of VEGF/GAPDH and HIF1A/GAPDH changed little up to 48h postmortem in tissue samples from the brain, kidney and lung except for a mild deviation of HIF1A in the myocardium. However, the status was different for EPO mRNA, with extraordinary stability for postmortem degradation and a marked postmortem time-dependent increase in the EPO/GAPDH ratio for all tissue samples. The present study suggested the potential for applying quantitative analyses of mRNA transcripts to autopsy materials and indicated the significance of investigating degradation profiles prior to carrying out relative quantification of target mRNAs in autopsy materials.

The hypophysis is embedded in the fossa at the base of skull, having important functions in the hormonal system. The present study investigated its postmortem morphological changes and the stability of adenohypopyseal hormones. The pituitaries were collected at autopsy 6 h to 20 days postmortem and were studied by histology, immunohistochemistry and electron microscopy. To avoid the influence of prolonged brain hypoxia or swelling, subjects who survived not longer than 12 h were examined. Histological changes were seen in the nucleus 6 h after death, followed by cytoplasmic changes, and the cell shapes were hardly identifiable 7 days postmortem. Electron microscopy revealed evident ultra-structural changes 6 h postmortem, involving rough endoplasmic reticulum, Golgi complexes, mitochondria, nuclei and cell membranes. However, secretory granules remained well preserved 7 days postmortem. Immunostaining showed positivities for growth hormone, prolactin, adenocorticotropic hormone, luteinizing hormone and thyroid-stimulating hormone up to 15 days after death. These findings suggest the usefulness of immunohistochemical investigation of the adenohypophysis for estimating the time of death and endocrinologic evaluation in decomposed cadavers.

In postmortem biochemistry, there is insufficient data available for the practical analysis of factors in the pericardial fluid. The aim of the present study was to examine postmortem pericardial fluid for urea nitrogen (UN), creatinine (Cr) and uric acid (UA) levels to investigate the pathophysiology of death in forensic autopsy cases (total, n = 409; within 48 h postmortem), which included blunt, sharp instrument injury, asphyxiation, drowning, fire fatalities, hyperthermia, hypothermia, methamphetamine-related fatalities, other poisoning, delayed death from trauma and natural diseases. There was a significant elevation in the three markers for chronic renal failure, gastrointestinal bleeding, hyperthermia, hypothermia, methamphetamine fatalities and delayed traumatic death, which was comparable with the clinical criteria for their serum levels. These postmortem findings showed azotemia due to renal failure, elevated protein catabolism and rhabdomyolysis. Although the pericardial levels were otherwise similar to the clinical serum reference ranges, only the drowning fatalities showed significantly lower levels for each marker. These observations suggested the stability of UN, Cr and UA in the pericardial fluid within 48 h postmortem and their usefulness for the pathophysiological investigation of death involving azotemia.

The aim of the present study was to examine the factors that contribute to the postmortem lung weight in acute fire fatalities (n=149) including those with lower (<60%) and higher (>60%) blood carboxyhemoglobin (COHb) levels (n=94 and 55, respectively). The control groups consisted of acute myocardial infarction/ischemia (AMI, n=99) and mechanical asphyxiation (n=85). For all cases (n=333), the lung and heart weights were independent of the postmortem time (4.5-72 h) and charring of the body. The combined weight of both lobes of the lung showed a significant gender difference (males>females, P<0.001), negative regression with respect to age (R=0.167, P<0.01) and positive regression with respect to heart weight (R=0.316, P<0.001). The gender difference was also significant for each cause of death even after being corrected using body height measurements. When the lung-heart weight ratio was estimated to diminish the influence of possible cardiogenic factors, the gender difference was insignificant for each cause of death among the non-elderly (<60 years of age). However, for elderly fire fatalities (>60 years of age), there was a gender difference (males>females) for the lower COHb group (P<0.05) and higher COHb group (P<0.001). A significant age-dependent difference (non-elderly>elderly) in the lung-heart weight ratio was observed for fire fatalities with a lower COHb and AMI among males and for fire fatalities with a higher COHb among females. Such gender- and/or age-dependent influences were not significant for fatal mechanical asphyxiation. These findings suggest that a person's heart weight may be a possible contributory factor to an increase in the lung weight in acute death, and that gender- and/or age-dependent susceptibilities may be additional factors that contribute to fire fatalities and AMI. In addition, elderly females appear to be most susceptible among fire casualties, and extreme cardiomegaly may also be a potential fatal risk factor.