Tomomi Michiue

Stability of endogenous reference genes in postmortem human brains for normalization of quantitative real-time PCR data: comprehensive evaluation using geNorm, NormFinder, and BestKeeper

In forensic molecular pathology, quantitative real-time polymerase chain reaction (RT-qPCR) provides a rapid and sensitive method to investigate functional changes in the death process. Accurate and reliable relative RT-qPCR requires ideal amplification efficiencies of target and reference genes. However, the amplification efficiency, changing during PCR, may be overestimated by the traditional standard curve method. No single gene meets the criteria of an ideal endogenous reference. Therefore, it is necessary to select suitable reference genes for specific requirements. The present study evaluated 32 potential reference genes in the human brain of 15 forensic autopsy cases using three different statistical algorithms, geNorm, NormFinder, and BestKeeper. On RT-qPCR data analyses using a completely objective and noise-resistant algorithm (Real-time PCR Miner), 24 genes met standard efficiency criteria. Validation of their stability and suitability as reference genes using geNorm suggested IPO8 and POLR2A as the most stable ones, and NormFinder indicated that IPO8 and POP4 had the highest expression stabilities, while BestKeeper highlighted ABL1 and ELF1 as reference genes with the least overall variation. Combining these three algorithms suggested the genes IPO8, POLR2A, and PES1 as stable endogenous references in RT-qPCR analysis of human brain samples, with YWHAZ, PPIA, HPRT1, and TBP being the least stable ones. These findings are inconsistent with those of previous studies. Moreover, the relative stability of target and reference genes remains unknown. These observations suggest that suitable reference genes should be selected on the basis of specific requirements, experiment conditions, and the characteristics of target genes in practical applications.

Postmortem biochemistry and immunohistochemistry of adrenocorticotropic hormone with special regard to fatal hypothermia

Adrenocorticotropic hormone (ACTH) is involved in systemic reactions to stress. The aim of the present study was a comprehensive analysis of serum and cerebrospinal fluid (CSF) levels of ACTH, and the pituitary immunohistochemistry with special regard to fatal hypothermia in routine forensic autopsy cases (n=162: 5-97 years of age; 114 males and 48 females; 4 h to 3 days postmortem, median, 19.2 h). The ACTH concentrations were independent of the postmortem time, gender, or age of the subjects. The serum ACTH level was similar to the clinical reference value for sharp instrument injury, fire fatality, and hypothermia, but was lower in other groups including hyperthermia, in particular for asphyxia and poisoning. The CSF level was usually much higher than the serum level, but was significantly lower for hypothermia and hyperthermia than in other groups (p<0.01). The rate of ACTH-immunopositivity in the anterior pituitary was low in cases of fatal hypothermia and hyperthermia, while it was high in cases of blunt injury, fire fatality, and acute ischemic heart disease. These observations showed that ACTH levels in the serum and CSF depended on the cause of death. The serum level was maintained despite a low CSF level and pituitary immunopositivity for fatal hypothermia, while the serum and CSF levels as well as pituitary immunopositivity were decreased for hyperthermia.

Immunohistochemistry of catecholamines in the hypothalamic–pituitary–adrenal system with special regard to fatal hypothermia and hyperthermia

Catecholamines are involved in various stress responses. Previous studies have suggested applicability of the postmortem blood levels to investigations of physical stress responses or toxic/hyperthermic neuronal dysfunction during death process. The present study investigated cellular immunopositivity for adrenaline (Adr), noradrenaline (Nad) and dopamine (DA) in the hypothalamus, adenohypophysis and adrenal medulla with special regard to fatal hypothermia (cold exposure) and hyperthermia (heat stroke) to examine forensic pathological significance. Medicolegal autopsy cases (n=290, within 3 days postmortem) were examined. The proportions of catecholamine (Adr, Nad and DA)-positive cells (% positivity) in each tissue were quantitatively estimated using immunostaining. Hyperthermia cases (n=12) showed a lower neuronal DA-immunopositivity in the hypothalamus than hypothermia cases (n=20), while Nad- and DA-immunopositivities in the adrenal medulla were higher for hyperthermia than for hypothermia. Rates of Nad-immunopositivity in the adrenal medulla were very low for hypothermia. No such difference between hypothermia and hyperthermia was seen in the adenohypophysis. In hypothermia cases, cellular Nad-immunopositivity in the adrenal medulla correlated with the Nad level in cerebrospinal fluid (r=0.591, p<0.01). These observations suggest a characteristic immunohistochemical pattern of systemic stress response to fatal hypothermia and hyperthermia, involving the hypothalamus and adrenal medulla.

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.

Creatine kinase-MB (CK-MB), cardiac troponin I (cTnI) and myoglobin (Mb) are biochemical markers of myocardial injury; however, Mb is more abundant in skeletal muscles. The present study involved analysis of these markers in pericardial and cerebrospinal fluids (PCF and CSF) from serial medicolegal autopsy cases (n=295, within 48h) to examine their efficacy in determining the cause of death. Although these markers showed a slight postmortem time-dependent elevation, except for CK-MB in CSF, the distribution depended on the cause of death. Mb levels in PCF and CSF were higher in fatal hyperthermia (heat stroke) and methamphetamine abuse, and CK-MB in both fluids was also higher in the latter. In psychotropic drug intoxication, CK-MB, cTnI and Mb were higher in PCF, but only cTnI was elevated in CSF. In electrocution and cerebrovascular disease, each marker was higher in PCF and also relatively high in CSF. PCF cTnI level was higher in acute pulmonary embolism without significant elevation of any other markers, whereas CSF CK-MB was higher in acute blunt brain injury death and methamphetamine abuse. In most cases of delayed brain injury death, hypothermia (cold exposure) and pneumonia, these markers were low or intermediate in both PCF and CSF; however, sudden cardiac death, asphyxiation and fire fatality cases showed few characteristic findings. These observations suggest that combined analyses of these markers in postmortem PCF and CSF, in addition to blood samples, are helpful for evaluating the severity of myocardial and/or skeletal muscle damage in death processes, in particular for investigating deaths due to hyperthermia, hypothermia, electrocution and intoxication.

Tissue-specific differences in mRNA quantification of glucose transporter 1 and vascular endothelial growth factor with special regard to death investigations of fatal injuries

Glucose transporter 1 (GLUT1) and vascular endothelial growth factor (VEGF) have been established as being responsible for cellular adaptation to oxygen deficiency in tissue ischemia and hypoxia mediated by hypoxia-inducible factor 1. We hypothesized that mRNA quantification of these factors in autopsy tissue specimens could have diagnostic significance for investigating the pathology of death, especially after injury. Various cases (total, n=119; less than 48h postmortem) were examined, including fatal blunt injury (n=71) and sharp instrument injury (n=18), as well as asphyxia (strangulation/hanging, n=12) and acute myocardial infarction/ischemia (n=18) as controls. Quantification of mRNA by TaqMan real-time RT-PCR and immunostaining were performed for GLUT1 and VEGF in lung, kidney, and skeletal muscle specimens. The postmortem interval showed no significant influence on the relative quantification of mRNA during the early postmortem period. Characteristic results were found in blunt injury cases: both GLUT1 and VEGF mRNAs decreased in the lung but increased in the skeletal muscle depending on survival time. In the kidney, subacute deaths showed higher GLUT1 mRNA levels compared with acute deaths from blunt injury, but no significant change was found for VEGF mRNA. Immunohistochemistry showed visually predominant GLUT1 immunoreactivity in the renal cortex for cases with a longer survival time, coincident with the results at the mRNA level. Tissue-specific differences in mRNA quantification of GLUT1 and VEGF shed light on tissue ischemia/hypoxia and subsequent tissue-dependent pathophysiological changes leading to death after injury.

Comparative evaluation of postmortem serum concentrations of neopterin and C-reactive protein

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.

Evaluation of postmortem serum and cerebrospinal fluid levels of thyroid-stimulating hormone with special regard to fatal hypothermia

The aim of the present study was to undertake, during routine forensic work, a comprehensive analysis of the serum and cerebrospinal fluid (CSF) levels of thyroid-stimulating hormone (TSH) and to examine hypophyseal TSH immunopositivity in relation to the cause of death, with particular regard to fatal hypothermia. Medicolegal autopsy cases (n=120; within 48 h postmortem; survival time, <24 h), including cases of blunt injury (n=9), sharp instrument injury (n=8), fire fatality (n=18), mechanical asphyxiation (n=10), drowning (n=21), poisoning (n=6), hypothermia (n=10), and acute ischemic heart disease (n=38), were examined. Serum and CSF TSH concentrations were measured using an electrochemiluminescence immunoassay. TSH immunoreactivity in adenohypophysis was quantitatively analyzed. Serum and CSF TSH levels were significantly lower in cases of hypothermia than in the other groups (p<0.05 and p<0.001, respectively). TSH immunopositivity in adenohypophysis was significantly lower in cases of hypothermia, but exhibited a large case-to-case variation for poisoning. These observations suggest that a decrease in serum and CSF TSH levels in hypothermia is related to hypothalamic adenohypophyseal dysfunction.

Postmortem Analyses of Drugs in Pericardial Fluid and Bone Marrow Aspirate

In forensic toxicology, bone marrow is often used when adequate blood samples are not available; however, pericardial fluid (PCF) has been poorly investigated. The present study comprehensively reviewed the toxicological data of blood, PCF and bone marrow aspirate (BMA) in forensic autopsy cases to investigate drug distribution. Analysis using automated gas chromatography/mass spectrometry (GC-MS) following solid/liquid phase extraction detected 36 drugs in 218 cases (8.0% among 2,724 cases examined). Drug distribution varied by drug as well as partly by case even when taken as a mixture. Most of the drugs showed overall similar distributions in right heart blood, PCF and BMA with some exceptions, however, several drugs, including phenothiazine derivatives and antidepressants, were detected at ∼1.5 times (1.2-2.0) higher levels in BMA than in right heart blood, but PCF levels were mostly equivalent to blood levels. Midazolam, propofol and thiamylal (intravenous anesthetics) were detected at a substantially lower concentration in PCF than in blood or BMA. These observations suggest that PCF and BMA are useful materials to be included in the forensic toxicological routine when blood samples are not available, as well as to investigate pharmaco-/toxicokinetics and postmortem redistribution.

Suicidal vehicle-assisted ligature strangulation resulting in complete decapitation: An autopsy report and a review of the literature

The victim (59-year-old male) used a long hemp rope tied between his neck and a cherry tree while attempting to drive his car away, resulting in complete decapitation. At autopsy, the decapitation wound of the head and the torso corresponded perfectly; a clear-cut severance plane was found at the bottom of the skull. In contrast to suicidal decapitation by hanging and traumatic railway injury, autopsy findings for vehicle-assisted ligature strangulation are rarely reported. A review of the literature concerning suicidal vehicle-assisted ligature strangulation suggested a striking young or adult male predominance, and the wound margins were usually clear-cut with a sharply-demarcated encircling abrasion zone. The present case presented some notable autopsy findings involving wound morphology and pathological changes in organs related to the mechanisms of injury and death. Despite complete decapitation, the face was congestive, the lungs were congested with findings of acute respiratory distress, and the brain was markedly swollen with diffuse and severe astrocyte injury, suggesting that asphyxiation was involved in the death before decapitation.

Postmortem catecholamine levels in pericardial and cerebrospinal fluids with regard to the cause of death in medicolegal autopsy

Previous studies have suggested that postmortem serum catecholamine levels reflect the magnitude of physical stress responses or toxic/hyperthermic neuronal dysfunction during the death process. The present study investigated postmortem adrenaline (Adr), noradrenaline (Nad), and dopamine (DA) levels in pericardial fluid (PCF) and cerebrospinal fluid (CSF) with regard to the cause of death, compared with right heart blood levels, in serial medicolegal autopsy cases with a postmortem time within 48 h (n=494). Correlations between PCF and CSF Adr levels, and those among right heart blood, PCF, and CSF DA levels were marked (r=0.66-0.83, p<0.0001), but were otherwise lower (r=0.22-0.44). With regard to the cause of death, Adr and Nad levels in PCF, CSF, and right heart blood mostly presented similar findings: these levels were generally high in injury, intoxication, and hyperthermia (heatstroke), but were low in hypothermia (cold exposure). DA levels at each site were higher in injury and intoxication. In addition, higher levels were detected for Nad levels in sharp instrument injury, as well as Adr, Nad, and DA in carbon monoxide intoxication at each site, and for CSF Nad in psychotropic drug intoxication. These findings suggest that characteristic elevations in Adr, Nad, and DA levels in PCF and CSF are involved in systemic responses to fatal stress and toxic neuronal dysfunction, reflecting the magnitude of such responses in individual cases.