Simple and simultaneous quantification of cyanide, ethanol, and 1-propanol in blood by headspace GC-MS/NPD with Deans switch dual detector system.

Cyanide is a powerful and rapidly acting poison. In Japan, cyanide poisoning is rare, and regular cyanide testing can be costly and time consuming. In contrast, alcohol analysis is routinely performed in most forensic laboratories. In this study, we attempted to develop a method for the simultaneous quantification of cyanide and alcohols in blood using headspace gas chromatography (HS-GC). As nitrogen-phosphorus detection (NPD) is more sensitive to hydrogen cyanide than mass spectrometry (MS), a Deans switch was used to switch the detectors during a single run. The separation provided by three analytical columns, PoraBOND Q, CP-Sil 5 CB, and HP-INNOWax, was investigated, and PoraBOND Q was selected. The use of HS-GC-MS/NPD with a Deans switch enabled the simple and simultaneous quantification of cyanide, ethanol, and 1-propanol. Eighteen other volatile compounds were detected in the SIM/scan mode of the MS.

The derivation of predictive equations to speculate the post-mortem interval using cases with over 20-mL pleural effusion: A preliminary study.

Often, pleural effusion is noted at autopsy when the cause of death is drowning or diseases such as heart, renal and liver failure. Several studies have established a correlation between the concentrations of electrolytes in pleural effusion and the post-mortem interval (PMI) or those concentrations and drowning site. The present study aims to investigate the relationship between the amount of pleural effusion, concentrations of electrolytes and total protein in pleural effusion, by integrated interpretation using various factors such as the deceased's gender, age, cause of death, drowning site, PMI and body temperature. We included 40 cadavers (26 male, 14 female) with >20-mL pleural effusion, which were categorised into four groups as follows: freshwater drowning; brackish water drowning; seawater drowning (drowning group); and not drowning. An equation derived to assess the lung weight revealed that the drowning site affected the lung weight. An equation for the amount of pleural effusion in the drowning group for the first time revealed that the amount of pleural effusion was directly proportional to the PMI. Using an equation to assess the PMI, we could estimate the PMI within 13.0-13.2 h in cases with >20-mL pleural effusion. Despite a small number of cases in the present study, we attained exciting results from the integrated statistical analysis.

Predictive equation for post-mortem interval using spectrophotometric values of post-mortem lividity: A pilot study.

The colour of post-mortem lividity and control skin of 86 cadavers was measured spectrophotometrically to obtain L* (value), a* (chroma) and b* (hue) values. In addition, left heart blood (n = 58), right heart blood (n = 57) and blood from the femoral vein (n = 21) were measured. Using these data, we analysed the relationship between post-mortem lividity, control skin and blood colours. L* of post-mortem lividity (L*p) and control skin (L*c) were strongly correlated ( r = 0.64). a* and b* of post-mortem lividity (a*p and b*p) significantly increased with an increasing post-mortem interval (PMI) but r2 values were low (0.11 and 0.070, respectively). Predictive equations for post-mortem lividity colour (L*p, a*p and b*p) were developed using control skin colour (L*c, a*c and b*c) and autopsy findings for the first time. The predictive equation for L*p explained almost 65% of the observed L*p. We created predictive equations for PMI with and without blood colour values, and the most accurate equation, which did not use blood colour values, made it possible to estimate PMI within ± 10.29 h. Further study of these equations will help us to understand the factors that affect post-mortem lividity colour and increase the accuracy of equations for predicting post-mortem lividity colour and PMI.

Predictive equation to estimate post-mortem interval using spectrophotometric blood-colour values.

Forensic pathologists use post-mortem phenomena to estimate the post-mortem interval (PMI). We have reported on the usefulness of post-mortem lividity spectrophotometric values to estimate PMIs. Here, we focused on blood colour, looking for associations between blood colour, age and PMI. We generated predictive equations for blood-colour values and the PMI. We included data from a total of 129 cadavers (84 males and 45 females). We measured the colour of 124 left ventricular blood ( L*l, a*l, b*l), 123 right ventricular blood ( L*r, a*r, b*r) and 57 femoral blood ( L*f, a*f, b*f) samples. We found no significant associations between blood colour and age or between blood colour and the PMI, but the values of a*l, b*l, a*r and b*r were significantly increased with increased age, and those of L*f, a*f and b*f were significantly decreased with increased PMI. We created equations to estimate blood colour. The equations for femoral blood colour had higher adjusted R2 values and lower root mean square error values than those for left and right ventricular blood colours. We generated equations to estimate PMIs using blood-colour values and autopsy findings. Our estimated PMIs up to 67 hours had accuracies within 8.84 hours, without measuring post-mortem lividity colour or considering the age of the deceased. This is the first study to estimate PMIs based on blood-colour spectrophotometric values.

Utilization effect of floor plan sketches in hypothetical cases assuming a community care meeting.

Objective　Floor plan sketches (FPSs) are schematic representations of floors in a home. FPSs display information gathered from observations and interviews on people's way of dwelling. To elucidate the effects of utilizing FPSs in case reviews assuming a community care meeting attended by multidisciplinary professionals, we conducted reviews of hypothetical cases created for experimental purposes.Methods　Two hypothetical cases (Cases 1 and 2) were developed, and each case was reviewed with and without FPSs. Two groups (Groups A and B) were created, each consisting of five health care and welfare professionals involved in actual case reviews. Group A reviewed Case 1 without FPSs followed by Case 2 with FPSs, while Group B reviewed Case 2 without FPSs followed by Case 1 with FPSs. Case conferences and group interviews conducted after the completion of these reviews. Based on the verbatim transcripts of the reviews and interviews, we investigated differences between case reviews with and without FPSs with regard to the time required for the review and the number and contents of participants' comments.Results　Review content could largely be divided into two categories: (1) the living conditions and support for the case subjects and their families, and (2) their homes and their way of dwelling at home. These categories were common to case reviews both with and without FPSs. In discussions about the homes and ways of dwelling, however, confirmation of the locations of rooms consumed a large amount of time in case reviews without FPSs. In case reviews with FPSs, discussions were more specific and included details such as room usage and paths by which residents move. The mean time required for a review was 41 minutes per case (range: 36 to 44 minutes), which did not greatly differ based on whether or not FPSs were used. Participants made more comments and seemed to interact more actively with each other when they had the FPSs than when they did not. The impressions of participants were that FPSs allowed the visualization of the case subjects and their families in their homes and fostered a greater feeling of familiarity with the case.Conclusion　The use of FPSs in case reviews reduces the time spent on information sharing and allows more detailed review contents. Furthermore, FPSs enhance the ability to imagine the daily lives of case subjects and their families, thereby potentially broadening assessments in case reviews.

Analysis of postmortem changes in internal organs and gases using computed tomography data.

Postmortem computed tomography (PMCT) is a useful method to identify various causes of death and measure the volume of internal organs and gases. The purpose of this study was to investigate postmortem changes as measured by PMCT, and the relationship between the volume of organs and gases and postmortem interval (PMI). Forty-six cadavers (22 men, 24 women) were examined by CT before autopsy. The volumes of the lungs, intrahepatic gas, and intrarectal gas were measured by CT using a workstation. A stepwise regression analysis was used to establish a predictive equation to ascertain the measured volume using factors including sex, age, height, body mass index (BMI), body surface area (BSA), and PMI. For estimation of PMI, stepwise regression analysis was used. In the equations for each measured volume, height, diaphragmatic height, and BSA were adopted for the left lung; height and diaphragmatic height were adopted for the right lung; PMI was adopted for intrahepatic gas; and sex and PMI were adopted for intrarectal gas. In the PMI equations, left lung volume, intrahepatic gas, and intrarectal gas were adopted together with sex, weight, and BMI. Values of intrahepatic gas decreased with postmortem changes, while intrarectal gas increased. This may be useful in investigation of postmortem changes. It will be necessary to include other parts of the intestine and to analyze volume changes in gases from these parts after death.

Expression of mRNA in the frontal cortex and hypothalamus in a rat model of acute carbon dioxide poisoning.

Acute carbon dioxide (CO2) poisoning causes no specific features that are revealed upon autopsy, and the pathophysiological mechanism of this syndrome is unclear. To address this issue, in the present study, we exposed rats to CO2 concentrations ranging from 10% to 60% and determined the effects on mRNA expression. According to the results of Gene Ontology (GO) and cluster analyses of microarrays data, we selected the following genes for further analysis: alkylglycerone phosphate synthase (Agps), hypocretin (Hcrt), tyrosine hydroxylase (Th), heat shock protein beta 2 (Hspb2), and opioid receptor delta 1 (Oprd1) expressed in the frontal cortex and renin (Ren), pancreatic polypeptide (Ppy), corticotropin releasing hormone receptor 2 (Crhr2), carbonic anhydrase 1 (Car1), and hypocretin receptor 1 (Hcrtr1) expressed in the hypothalamus. We found significant differences between the expression levels of Agps and Hspb2 mRNAs in the frontal cortex and that of Ppy, Crhr2 mRNAs in the hypothalamus in the presence of high concentrations of CO2. Further investigation of these genes may clarify the pathophysiology of acute CO2 poisoning and facilitate the development of novel forensic tests that can diagnose the cause of death.

[An Autopsy Case of Abnormal Behaviour Induced by Zolpidem].

Zolpidem is a widely used ultrashort-acting non-benzodiazepine in clinical practice; compared with benzodiazepines, it does not have side effects such as daytime hangover, rebound insomnia, and development of tolerance. We report an autopsy case of abnormal behaviour induced by zolpidem. A man in his 60's had suffered from postherpetic neuralgia about 2 months ago and had been prescribed zolpidem for insomnia. According to his family, he had no memory of his actions such as striking a wall, taking his futon outside, and eating 5 times a day after he took zolpidem. Because his postherpetic neuralgia did not improve, he was hospitalized and treated with an epidural block. During hospitalization, he took off his clothes, removed the epidural block catheter by himself, and slept on others' beds. He disappeared from the hospital one day; the next day, he was found dead in a narrow water storage tank 10 km away from the hospital. He was thought to have driven a car by himself to reach the place. Forensic autopsy revealed that the cause of death was drowning. Zolpidem and several other drugs were detected by toxicological analysis of his blood; the concentrations of these drugs were within therapeutic range. There are several reports about somnambulism induced by zolpidem such as sleepwalking, sleep driving, and eating. Considering the strange episodes following zolpidem administration, his behaviour on the day of his death was considered abnormal behaviour induced by zolpidem.

A fatal case of poisoning related to new cathinone designer drugs, 4-methoxy PV8, PV9, and 4-methoxy PV9, and a dissociative agent, diphenidine.

A woman in her thirties was found dead on a bed. Considerable amounts of "aroma liquid" and "bath salt" products and hypnotic drug tablets were scattered beside the bed. Autopsy showed pulmonary congestion and edema. Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) analyses of "aroma liquid" and "bath salt" products showed the presence of new cathinone designer drugs, 4-methoxy PV8 (4-methoxy PHPP), PV9 (α-POP), and 4-methoxy PV9 (4-methoxy α-POP), and a dissociative agent, diphenidine. Drug screening in stomach contents, blood and hydrolyzed urine of the woman by GC-MS and liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed the presence of the above 4 types of drugs and 3 types of benzodiazepines, triazolam, flunitrazepam, and nitrazepam, and their metabolites. The above 7 drugs and 3 benzodiazepine metabolites were simultaneously determined by LC-MS/MS after modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) extraction using diazepam-d5 as the internal standard. The concentrations of 4-methoxy PV8, PV9, 4-methoxy PV9, and diphenidine in the femoral blood were 2.69, 0.743, 0.261, and 1.38µg/ml, respectively, which were significantly higher than concentrations reported in previous cases. Alcohol concentration in the femoral blood was 1.52mg/ml. Based on the pathological and toxicological findings, the cause of death was determined to be 3 types of cathinone drugs, 4-methoxy PV8, PV9, and 4-methoxy PV9, and diphenidine poisoning under the influence of 3 benzodiazepines and alcohol.

Relationship between T-cell HLA-DR expression and intravenous immunoglobulin treatment response in Kawasaki disease.

BACKGROUND: Kawasaki disease (KD) is an acute febrile illness associated with the development of vasculitis. Administration of intravenous immunoglobulin (IVIG) is the standard treatment for KD. However, IVIG treatment is not effective in approximately 15% of children with KD. Some reports have presented evidence of immunological responses in IVIG-resistant KD patients. We assessed the possibility that T-cell activation is a contributing mechanism underlying this phenomenon. METHODS: We analyzed human leukocyte antigen-DR (HLA-DR) expression on peripheral blood CD4+ and CD8+ T cells in 82 children with KD who were admitted to the hospital between October 2007 and February 2012. We compared the percentages of HLA-DR+ T cells among the CD4+ T-cell and CD8+ T-cell populations for the IVIG-effective and IVIG-resistant groups. RESULTS: Among the 82 subjects, 51 had IVIG-effective KD and 31 children had IVIG-resistant KD. The percentages of HLA-DR+ T cells among the CD4+ T-cell and CD8+ T-cell populations in the IVIG-effective group were significantly lower than those in the IVIG-resistant group. CONCLUSION: Our results suggest that increased T-cell HLA-DR expression is associated with IVIG resistance in KD patients, indicating that HLA-DR expression would be a useful tool for predicting IVIG responsiveness during KD pathogenesis.

Anomalous origin of the right coronary artery from the left coronary sinus with an intramural course: comparison between sudden-death and non-sudden-death cases.

BACKGROUND: The prognosis of patients in whom the right coronary artery (RCA) arises from the left coronary sinus (LCS) is unequal. An initial intramural course of the coronary artery within the aortic media is considered to cause myocardial ischemia in cases of coronary anomalies. METHODS: Clinicopathological findings in five autopsy cases where the RCA arose from the LCS with an intramural course were examined. Comparison between sudden cardiac death and noncardiac death was also performed. RESULTS: Two of five cases were sudden cardiac death, and the other three cases were noncardiac death. In one case of sudden death, the person collapsed during light exercise, and in the other case, the person was under the effect of methamphetamine. Both of the cases of sudden death showed an RCA-dominant pattern in distribution of the coronary artery, atherosclerotic narrowing of the RCA, and acute ischemic necrosis in the posterior basilar ventricular septum around the atrioventricular conduction system, which is considered to be the territory of the RCA. CONCLUSIONS: An intramural course within the aortic media may be an accelerating factor of decreased blood flow in cases with an origin of the RCA arising from the LCS because of compression from the aortic lumen. However, this finding may not be an independent predictor of pathological ischemia. Additional factors that diminish blood flow in the intramural segment may be required to cause significant myocardial ischemia. Additionally, inciting factors, which can increase blood pressure, may also play a role in causing symptomatic myocardial ischemia by initiating mechanical compression from the aorta to the intramural segment of the RCA.

Serum levels of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinases-1 in human herpesvirus-6-infected infants with or without febrile seizures.

Human herpesvirus-6 (HHV-6) is a cause of exanthema subitum and, sometimes, of febrile seizures. However, the pathogenesis of febrile seizures associated with HHV-6 infection remains unclear. We investigated serum matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinases-1 (TIMP-1) levels in infants with HHV-6 infection. Serum levels of both MMP-9 and TIMP-1 were significantly higher in infants with HHV-6 infection than in controls. Serum TIMP-1 levels were significantly higher in infants with febrile seizures than in infants without febrile seizures. Serum MMP-9/TIMP-1 ratios were significantly lower in infants with febrile seizures than in infants without febrile seizures. In infants with HHV-6 infection, positive correlations were found between serum MMP-9 concentrations and the white blood cells (WBC) count, and between serum TIMP-1 concentrations and the WBC count. Positive correlations were also found between the amounts of HHV-6 DNA and the ratios of MMP-9/TIMP-1 in infants with HHV-6 infection. In conclusion, we suggest that high serum levels of MMP-9 and TIMP-1 in infants with HHV-6 infection may induce dysfunction of the blood-brain barrier, eventually causing febrile seizures.

Medical neglect death due to acute lymphoblastic leukaemia: an autopsy case report.

We report the case of 2-year-old girl who died of precursor B-cell acute lymphoblastic leukaemia (ALL), the most common cancer in children. She had no remarkable medical history. She was transferred to a hospital because of respiratory distress and died 4 hours after arrival. Two weeks before death, she had a fever of 39 degrees C, which subsided after the administration of a naturopathic herbal remedy. She developed jaundice 1 week before death, and her condition worsened on the day of death. Laboratory test results on admission showed a markedly elevated white blood cell count. Accordingly, the cause of death was suspected to be acute leukaemia. Forensic autopsy revealed the cause of death to be precursor B-cell ALL. With advancements in medical technology, the 5-year survival rate of children with ALL is nearly 90%. However, in this case, the deceased's parents preferred complementary and alternative medicine (i.e., naturopathy) to evidence-based medicine and had not taken her to a hospital for a medical check-up or immunisation since she was an infant. Thus, if she had received routine medical care, she would have a more than 60% chance of being alive 5 years after diagnosis. Therefore, we conclude that the parents should be accused of medical neglect regardless of their motives.

Relationship between the matrix effect and the physicochemical properties of analytes in gas chromatography.

The phenomenon "matrix-induced chromatographic response enhancement" (matrix effect) causes quantitative errors in gas chromatography (GC) analyses. This effect varies according to the analyte nature, matrix type and concentration, and GC-system parameters. By focusing on the physicochemical properties of analytes, a predictive model was developed for the matrix effect using quantitative structure-property relationships. Experimental values of the matrix effect were determined for 58 compounds in a serum extract obtained from solid-phase extraction as the matrix. Eight molecular descriptors were selected, and the matrix-effect model was developed by multiple linear regression. The developed model predicted values for the matrix effect without any further experimental measurements. It also indicated that the molecular polarity (particularly H-bond donors) and volume of the analyte increase the matrix effect, while hydrophobicity and increasing number of nonpolar carbon atoms in the analyte decrease the matrix effect. The model was applied to the analysis of barbiturates. The predicted values indicated that N-methylation decreases the matrix effect, and the relative predicted values were effective for the selection of an internal standard. The obtained insight into the matrix effect and the prediction data will be helpful for developing quantitative analysis strategies.

A familial case of ABO phenotype-genotype discrepancy.

In the present study, we detected a familial case of ABO phenotype-genotype discrepancy. Although the observed phenotypes were B and O, the corresponding ABO genotypes were AB and AOG, respectively, by routine examination. Exons 6 and 7 of the ABO gene were sequenced and subsequently, the sequence of the allele responsible for ABO phenotype-genotype discrepancy was examined. Our results indicated that the Ax allele was present in 3 family members. By employing traditional serological methods, we further identified and confirmed that the allele Ax and the resulting Ax phenotype are responsible for ABO phenotype-genotype discrepancy.

1α,25-Dihydroxyvitamin D(3) inhibits vascular cellular adhesion molecule-1 expression and interleukin-8 production in human coronary arterial endothelial cells.

Kawasaki disease is an acute febrile vasculitis of childhood that is associated with elevated production of inflammatory cytokines, causing damage to the coronary arteries. The production of proinflammatory cytokines and expression of adhesion molecules in human coronary arterial endothelial cells (HCAECs) is regulated by nuclear transcription factor-κB (NF-κB) activation. We have previously reported that the active form of vitamin D, 1α,25-dihydroxyvitamin D(3) (1α,25-(OH)(2)D(3)), inhibits tumor necrosis factor-α (TNF-α)-induced NF-κB activation. In this study, we examined the anti-inflammatory effects of 1α,25-(OH)(2)D(3) on TNF-α-induced adhesion molecule expression (vascular cellular adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1)) and cytokine production (interleukin-6 (IL-6) and IL-8) in HCAECs. Pretreatment with 1α,25-(OH)(2)D(3) significantly inhibited TNF-α-induced VCAM-1 expression and IL-8 production in HCAECs. Our results suggest that adjunctive 1α,25-(OH)(2)D(3) therapy may modulate the inflammatory response during Kawasaki disease vasculitis.

Rapid and reliable screening method for detection of 70 pesticides in whole blood by gas chromatography-mass spectrometry using a constructed calibration-locking database.

Pesticide poisoning is one of the most common causes of death by poisoning in Japan, and various kinds of pesticides including organophosphates, carbamates and pyrethroids are listed as causative substances. The purpose of our study was to develop a rapid and reliable screening method for various kinds of pesticides in whole blood by using a unique calibration-locking database and gas chromatography-mass spectrometry. A database of 70 pesticides was constructed using NAGINATA™ software with parameters such as mass spectrum, retention time and qualifier ion/target ion ratio (QT ratio) and calibration curve. Diazepam-d(5) was used as the internal standard for construction of each calibration curve within the range of 0.01-5.0 µg/ml. We examined the applicability of the constructed database by analyzing whole blood samples spiked with 70 pesticides. The pesticides in blood were extracted with hexane under acidic conditions or with an enhanced polymer column (Focus™), subjected to GC-MS, and screened by the pesticides database. Among the 70 pesticides examined, 66 and 62 were successfully identified at the level of 1 and 0.1 µg/ml, respectively, by hexane and 63 and 51 were identified by the Focus column without the use of standard compounds. The time required for data analysis was significantly reduced. Since the established method can produce qualitative and semi-quantitative data without the need for standard substances, this new screening method using NAGINATA™ should be useful for confirming the presence of pesticides in blood in future clinical and forensic cases.

Molecular epidemiological study of human rhinovirus species A, B and C from patients with acute respiratory illnesses in Japan.

Recent studies suggest that human rhinovirus species A, B and C (HRV-ABCs) may be associated with both the common cold and severe acute respiratory illnesses (ARIs) such as bronchiolitis, wheezy bronchiolitis and pneumonia. However, the state and molecular epidemiology of these viruses in Japan is not fully understood. This study detected the genomes of HRV-ABCs from Japanese patients (92 cases, 0-36 years old, mean±sd 3.5±5.0 years) with various ARIs including upper respiratory infection, bronchiolitis, wheezy bronchiolitis, croup and pneumonia between January and December 2010. HRV-ABCs were provisionally type assigned from the pairwise distances among the strains. On phylogenetic trees based on the nucleotide sequences of the VP4/VP2 coding region, HRV-A, -B and -C were provisionally assigned to 14, 2 and 12 types, respectively. The present HRV-A and -C strains had a wide genetic diversity (>30 % divergence). The interspecies distances were 0.230±0.063 (mean±sd, HRV-A), 0.218±0.048 (HRV-B) and 0.281±0.105 (HRV-C), based on nucleotide sequences, and 0.075±0.036 (HRV-A), 0.049±0.022 (HRV-B) and 0.141±0.064 (HRV-C) at the deduced amino acid level. Furthermore, HRV-A and -C were the predominant species and were detected throughout the seasons. The results suggested that HRV-A and -C strains have a wide genetic divergence and are associated with various ARIs in Japan.

An autopsy case of misdiagnosis based on postmortem computed tomography findings.

A middle-aged man was found lying beside his bicycle on an early winter morning. The cause of death was diagnosed by clinicians as traumatic intracerebral hemorrhage and cerebral contusion with frontal bone fracture based on the findings of Computed Tomography (CT) of the head. However, forensic autopsy revealed that there were no evidences of intracerebral hemorrhage and left frontal bone fracture but the defect of golf ball size on the frontal lobe which was considered to be a complication from the old cerebral contusion and old bone fracture. The bleeding and pooling blood from subarachnoid hemorrhage (SAH) to the frontal lobe defect had the appearance of an intracerebral hemorrhage. Disruption of left renal artery was found and the cause of death was diagnosed as massive hemorrhage due to this rupture. Although postmortem CT is a useful tool for obtaining information on the body prior to conducting an autopsy, it should be used with extreme caution.