Establishment of widely applicable DNA extraction methods to identify the origins of crude drugs derived from animals using molecular techniques.

We established widely applicable DNA extraction methods to identify the origins of crude drugs derived from animals. Twenty-one samples including 17 kinds of crude drug derived from animals were examined. DNA was extracted from most of the crude drugs by adjustment of the QIAamp® DNA Mini Kit. DNA extraction was performed successfully using phenol to remove impurities after applying a proteinase treatment. DNA extraction was performed successfully by decalcification treatment using ethylenediaminetetraacetic acid (EDTA), before applying the proteinase treatment for crude drugs having high calcium content, such as those from oyster shell and cuttlefish bone. DNA could not be extracted from sea-ear shell using the EDTA decalcification treatment, but was extracted successfully using a TBONE EX KIT. The mitochondrial 16S ribosomal RNA (rRNA) gene region was amplified, and Basic Local Alignment Search Tool (BLAST) analysis was performed after sequencing. Polymerase chain reaction (PCR) products of approximately 600 bp in length were obtained from all samples except donkey glue, one of the two seahorses, and longgu. Drug origins were determined in all samples by sequence analysis based on the BLAST results, and match rates were >97 %. Moreover, 16 samples had a match rate >99 %. Our DNA extraction methods were widely applicable to evaluation of many crude drugs derived from animals, and proved very useful for identifying the origins of such drugs.

Distinction between saltwater drowning and freshwater drowning by assessment of sinus fluid on post-mortem computed tomography.

OBJECTIVE: To evaluate the difference in sinus fluid volume and density between saltwater and freshwater drowning and diagnose saltwater drowning in distinction from freshwater drowning. METHODS: Ninety-three drowning cases (22 saltwater and 71 freshwater) were retrospectively investigated; all had undergone post-mortem CT and forensic autopsy. Sinus fluid volume and density were calculated using a 3D-DICOM workstation, and differences were evaluated. Diagnostic performance of these indicators for saltwater drowning was evaluated using a cut-off value calculated by receiver operating characteristic (ROC) analysis. RESULTS: The median sinus fluid volume was 5.68 mL in cases of saltwater drowning (range 0.08 to 37.55) and 5.46 mL in cases of freshwater drowning (0.02 to 27.68), and the average densities were 47.28 (14.26 to 75.98) HU and 32.56 (-14.38 to 77.43) HU, respectively. While sinus volume did not differ significantly (p = 0.6000), sinus density was significantly higher in saltwater than freshwater drowning cases (p = 0.0002). ROC analysis for diagnosis of saltwater drowning determined the cut-off value as 37.77 HU, with a sensitivity of 77 %, specificity of 72 %, PPV of 46 % and NPV of 91 %. CONCLUSION: The average density of sinus fluid in cases of saltwater drowning was significantly higher than in freshwater drowning cases; there was no significant difference in the sinus fluid volume. KEY POINTS: • Sinus fluid density of saltwater drowning is significantly higher than freshwater drowning. • Cut-off value was 37.77 HU based on the ROC analysis. • The cut-off value translated to 91 % NPV for diagnosis of saltwater drowning.

An autopsy case of death due to metabolic acidosis after citric acid ingestion.

A man in his 40s was found unconscious on a sofa in a communal residence for people with various disabilities. He appeared to have drunk 800 ml of undiluted citric acid from a commercial plastic bottle. The instructions on the label of the beverage specified that the beverage be diluted 20- to 30-fold before consumption. The patient was admitted to an emergency hospital with severe metabolic acidosis (pH, 6.70; HCO3(-), 3.6 mEq/L) and a low ionized calcium level (0.73 mmol/L). Although ionized calcium and catecholamines were continuously administered intravenously to correct the acidosis, the state of acidemia and low blood pressure did not improve, and he died 20 h later. Citric acid concentrations in the patient's serum drawn shortly after treatment in the hospital and from the heart at autopsy were 80.6 mg/ml and 39.8 mg/dl, respectively (normal range: 1.3-2.6 mg/dl). Autopsy revealed black discoloration of the mucosal surface of the esophagus. Microscopically, degenerated epithelium and neutrophilic infiltration in the muscle layer were observed. In daily life, drinking a large amount of concentrated citric acid beverage is rare as a cause of lethal poisoning. However, persons with mental disorders such as dementia may mistakenly drink detergent or concentrated fluids, as in our case. Family members or facility staff in the home or nursing facility must bear in mind that they should not leave such bottles in places where they are easily accessible to mentally handicapped persons.

Post-mortem computed tomography findings of the lungs: Retrospective review and comparison with autopsy results of 30 infant cases.

OBJECTIVES: Infant cases frequently show a diffuse increase in the concentration of lung fields on post-mortem computed tomography (PMCT). However, the lungs often show simply atelectasis at autopsy in the absence of any other abnormal changes. Thus, we retrospectively reviewed the PMCT findings of lungs following sudden infant death and correlated them with the autopsy results. MATERIALS AND METHODS: We retrospectively reviewed infant cases (0 year) who had undergone PMCT and a forensic autopsy at our institution between May 2009 and June 2013. Lung opacities were classified according to their type; consolidation, ground-glass opacity and mixed, as well as distribution; bilateral diffuse and areas of sparing. Statistical analysis was performed to assess the relationships among lung opacities, causes of death and resuscitation attempt. RESULTS: Thirty infant cases were selected, which included 22 sudden and unexplained deaths and 8 other causes of death. Resuscitation was attempted in 22 of 30 cases. Bilateral diffuse opacities were observed in 21 of the 30 cases. Of the 21 cases, 18 were sudden and unexplained deaths. Areas of sparing were observed in 4 sudden and unexplained deaths and 5 other causes of death. Distribution of opacities was not significantly associated with causes of death or resuscitation attempt. The 21 cases with bilateral diffuse opacities included 6 consolidations (4 sudden and unexplained deaths, 2 other causes of death), 4 ground-glass opacities (3 sudden and unexplained deaths and 1 other) and 11 mixed (11 sudden and unexplained deaths). Types of opacities were not significantly associated with causes of death or resuscitation attempt. CONCLUSION: Atelectasis is very common in sudden and unexplained death of infants. Bilateral diffuse mixed opacity was observed only in sudden and unexplained deaths. Bilateral diffuse pure consolidation or ground-glass opacity was also observed in other causes of death.

Sex determination of the pelvis using Fourier analysis of postmortem CT images.

BACKGROUND: The Fourier transform (FT) is an image-processing tool that can be used to analyze space or linear systems. We used the FT to analyze the shape of the subpubic line (SL) and the right greater sciatic notch area line (GSNAL) of the pelvis to identify sexing in computed tomographic (CT) images. METHODS: 120 bodies (60 male, 60 female) were scanned by multislice CT before autopsy. SL and GSNAL images were reconstructed using an image workstation. The range of the SL is between the bottom ends of the bilateral acetabulum, and the range of the right GSNAL is from the posterior superior iliac spine to the ischial tuberosity. Coordinates (x, y) of the bone outline were extracted from CT images and calculated by fast FT. The Mann-Whitney U test comparing the amplitude of each frequency was used to determine significant differences between the two populations. Multiple regression analyses were performed to find statistically significant amplitudes of each frequency. We verified the accuracy of prediction of the regression equation, and a Cohen's kappa coefficient was measured. RESULTS: Statistically significant differences of amplitude were found in the 2nd, 3rd, and 4th frequencies of the SL and the 1st, 3rd, and 4th frequencies of GSNAL. Adjusted R(2) using all amplitudes having a significant difference was 0.772. The accuracy was 0.983. Kappa coefficient was 0.983. CONCLUSION: We could determine sexing from SL and GSNAL of the pelvis using FT with high accuracy. FT is a useful tool to analyze the curved shape of human bone.

Sudden death due to a cystic lesion in the cerebellum.

A middle-aged female patient with a depressive disorder presented to a mental hospital because of a 2-month worsening history of headache, dizziness, and nausea. The next morning, she was observed to be sleeping, but was then found dead 1h later. Postmortem computed tomography and autopsy revealed a large cyst in the right cerebellar hemisphere, hydrocephalus, and transforaminal herniation. Careful observation revealed an approximately 0.4cm×0.8cm slightly grayish discoloration in the cyst wall that was diagnosed as hemangioblastoma based on its histological features. Finally, we concluded that the cause of death in this case was attributable to the brain stem compression, which was caused by obstructive hydrocephalus secondary to the cystic hemangioblastoma in the cerebellum. The symptoms for 2 months before her death had most likely resulted from increased intracranial pressure. Hemangioblastomas usually appear as nodules in the wall of the cyst, but the tumor in our case looked like just a slightly grayish discoloration. Therefore, cystic lesions in the CNS need to be carefully examined.

Quantitative analysis of 3,4-dimethylmethcathinone in blood and urine by liquid chromatography-tandem mass spectrometry in a fatal case.

We report here the quantitative analysis of cathinone-type designer drug 3,4-dimethylmethcathinone (3,4-DMMC) in blood and urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a fatal case. Abuse of 3,4-DMMC is widespread and a global issue. However, to date, there have been no reports of 3,4-DMMC-related deaths. We encountered a death in which 3,4-DMMC was thought to play a causative role, and successfully identified this designer drug from biological samples by using LC-MS/MS and QuEChERS (quick, easy, cheap, effective, rugged and safe) extraction method. For standard samples, detection of 3,4-DMMC in human blood and urine samples in the calibration range (5-400 ng/mL) was successful with recoveries of 85.9-89.4% (blood) and 95.8-101% (urine), limits of detection of 1.03 (blood) and 1.37 ng/mL (urine) and limits of quantification of 5.00 (blood) and 5.38 ng/mL (urine). The concentrations of 3,4-DMMC in blood (external iliac vein) and urine in the case were 27 mg/L and 7.6 mg/L, respectively. Some metabolites, including 3,4-dimethylcathione (DMC) and ß-ketone reduced metabolites (ß-OH-DMMC and ß-OH-DMC), were detected in both blood and urine.

Sudden, unexpected infant death due to pulmonary arterial hypertension.

A 3-year-old girl with no particular medical history complained of a stomachache and died on the way to the hospital. The autopsy revealed marked right ventricular hypertrophy and dilation with no other cardiac abnormalities. Microscopically, the pulmonary small arteries showed marked medial hypertrophy and varying degrees of intimal and adventitial thickening. We supposed that the cause of death was attributable to pulmonary arterial hypertension (PAH). PAH is a rare disease that can cause sudden, unexpected death at any age. Forensic pathologists should consider PAH in the differential diagnosis of sudden death.

Diagnosis of drowning using post-mortem computed tomography based on the volume and density of fluid accumulation in the maxillary and sphenoid sinuses.

Recent studies have reported that drowning victims frequently have fluid accumulation in the paranasal sinuses, most notably the maxillary and sphenoid sinuses. However, in our previous study, many non-drowning victims also had fluid accumulation in the sinuses. Therefore, we evaluated the qualitative difference in fluid accumulation between drowning and non-drowning cases in the present study. Thirty-eight drowning and 73 non-drowning cases were investigated retrospectively. The fluid volume and density of each case were calculated using a DICOM workstation. The drowning cases were compared with the non-drowning cases using the Mann-Whitney U-test because the data showed non-normal distribution. The median fluid volume was 1.82 (range 0.02-11.7) ml in the drowning cases and 0.49 (0.03-8.7) ml in the non-drowning cases, and the median fluid density was 22 (-14 to 66) and 39 (-65 to 77) HU, respectively. Both volume and density differed significantly between the drowning and non-drowning cases (p=0.001, p=0.0007). Regarding cut-off levels in the ROC analysis, the points on the ROC curve closest (0, 1) were 1.03ml (sensitivity 68%, specificity 68%, PPV 53%, NPV 81%) and 27.5 HU (61%, 70%, 51%, 77%). The Youden indices were 1.03ml and 37.8 HU (84%, 51%, 47%, 86%). When the cut-off level was set at 1.03ml and 27.5HU, the sensitivity was 42%, specificity 45%, PPV 29% and NPV 60%. When the cut-off level was set at 1.03ml and 37.8HU, sensitivity was 58%, specificity 32%, PPV 31% and NPV 59%.

Postmortem radiography of gastromalacia: case reports.

Gastromalacia is a postmortem artifact resulting from autolysis of the gastric walls. Gastromalacia is autolytic rupture of the stomach caused by endogenous enzymes, and it is devoid of any vital reactions. The left leaf of the diaphragm is occasionally perforated by a ragged fenestration, with escape of gastric contents into the pleural cavity. This rupture may lead to pneumoperitoneum or pneumothorax. For diagnostic radiologists, gastromalacia is rarely encountered. Therefore, they should be aware of this entity to avoid misdiagnosis when performing postmortem radiography.

Hypothermic death: possibility of diagnosis by post-mortem computed tomography.

Referring to our experience with post-mortem computed tomography (CT), many hypothermic death cases presented a lack of increase in lung-field concentration, blood clotting in the heart, thoracic aorta or pulmonary artery, and urine retention in the bladder. Thus we evaluated the diagnostic performance of post-mortem CT on hypothermic death based on the above-mentioned three findings. Twenty-four hypothermic death subjects and 53 non-hypothermic death subjects were examined. Two radiologists assessed the presence or lack of an increase in lung-field concentration, blood clotting in the heart, thoracic aorta or pulmonary artery, and measured urine volume in the bladder. Pearson's chi-square test and Mann-Whitney U-test were used to assess the relationship between the three findings and hypothermic death. The sensitivity, specificity, accuracy, positive predictive value (PPV) and negative predictive value (NPV) of the diagnosis were also calculated. Lack of an increase in lung-field concentration and blood clotting in the heart, thoracic aorta or pulmonary artery were significantly associated with hypothermic death (p=0.0007, p<0.0001, respectively). The hypothermic death cases had significantly more urine in the bladder than the non-hypothermic death cases (p=0.0011). Regarding the diagnostic performance with all three findings, the sensitivity was 29.2% but the specificity was 100%. These three findings were more common in hypothermic death cases. Although the sensitivity was low, these findings will assist forensic physicians in diagnosing hypothermic death since the specificity was high.

Unexpectedly high blood concentration of bisoprolol after an incorrect prescription: A case report.

An elderly person died of uncontrolled bradycardia in a hospital. The doctor had prescribed 1.35 mg of bisoprolol fumarate orally, but a nurse mistakenly gave the patient 10 mg of the drug 9 hours prior to her death. Bisoprolol was detected in her blood by liquid chromatography-mass spectrometry at a concentration of 176 ng/mL. Even if the patient had chronic heart failure, this concentration is double the expected value. This patient was found to have a mutation within cytochrome P2D6, with thymidine substituted for cytosine at position 100 and cytosine for guanine at position 4180, causing proline to serine and threonine to serine amino acid substitutions. This mutation in the intermediate metabolizer allele reportedly reduces enzyme activity by half. However, in addition to the type of cytochrome P450 allelic variant, the amount of enzyme product influences metabolism of this drug. In this case, the high blood concentration of bisoprolol was only partly attributable to an error in prescription; its concentration was inexplicably high.

Assessment of the relationship between drowning and fluid accumulation in the paranasal sinuses on post-mortem computed tomography.

Recent reports have detailed common computed tomography (CT) findings in drowning victims, most notably fluid accumulation in the maxillary and sphenoidal sinuses. This CT finding could help forensic doctors to diagnose drowning. This study retrospectively investigated 151 subjects: 39 drowning and 112 non-drowning cases. Pearson's chi-square tests demonstrated that fluid accumulation in the maxillary or sphenoidal sinuses was associated significantly with drowning (p=0.0001). The sensitivity of the drowning diagnosis was 97%, specificity was 35%, accuracy was 51%, positive predictive value was 34% and negative predictive value was 98%. Drowning was significantly associated with fluid accumulation in the maxillary and sphenoidal sinuses, but the specificity and positive predictive value of the drowning diagnosis were poor. Although the presence of fluid in the maxillary and sphenoidal sinuses cannot be used to diagnose drowning, the absence of the fluid can be used to virtually exclude drowning.

Rapid determination of disulfoton and its oxidative metabolites in human whole blood and urine using QuEChERS extraction and liquid chromatography-tandem mass spectrometry.

A liquid chromatography-tandem mass spectrometry method was developed and validated for simultaneous determination of disulfoton and five of its oxidative metabolites (disulfoton-sulfoxide, disulfoton-sulfone, demeton-S, demeton-S-sulfoxide and demeton-S-sulfone) in human whole blood and urine. Extraction was undertaken using a QuEChERS method, which is commonly used in food analysis. D10-Disulfoton was used as the internal standard. Separation was carried out using a CAPCELL-PAK MG II column (35×2.0 mm i.d., 5 µm, Shiseido) with a mobile phase of 10 m mol/L ammonium formate and methanol. This method was applied in an autopsy case, and disulfoton and its oxidative metabolites were successfully detected in both blood and urine. The concentrations of disulfoton in the blood and urine were 360 and 23.8 ng/mL, respectively. There was a relatively low concentration of demeton-S in both the blood (4.0 ng/mL) and urine (45.7 ng/mL). To date, there have been no reported cases of detection of demeton-S in human samples.

Rapid drug extraction from human whole blood using a modified QuEChERS extraction method.

A modified QuEChERS (quick, easy, cheap, effective, rugged and safe) extraction method followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for the simultaneous determination of forensically important drugs and poisons (more than 90 compounds) in human whole blood. Because the QuEChERS method is commonly used for the analysis of pesticide residues in foods, we customized the QuEChERS method for forensic use. This extraction method consists essentially of two steps: (1) extraction/partitioning and (2) dispersive-solid phase extraction. In step 1, three-fold diluted blood was mixed with an internal standard (D5-diazepam for basic drugs or D5-phenobarbital for acidic drugs) solution, a QuEChERS pre-packed extraction kit (containing magnesium sulfate and sodium acetate) and a stainless steel bead, then partitioned into three layers by centrifugation. In step 2, the top layer (acetonitrile) was transferred into a centrifuge tube containing a dispersive-solid phase sorbent (containing primary secondary amine, end-capped octadecylsilane, and magnesium sulfate) and mixed for purification. After the centrifugation, supernatant was injected into LC-MS/MS. The QuEChERS method was applied in an autopsy case and we confirmed that this method can easily extract various types of drugs and metabolites from human whole blood. The combination of the modified QuEChERS method and LC-MS/MS could enable technicians inexperienced in forensic toxicological analysis to acquire reliable data quickly and easily.

Usefulness of postmortem computed tomography before forensic autopsy for alerting forensic personnel to tuberculosis infection.

Since May 2009, we have performed multislice computed tomography (MSCT) prior to forensic autopsy for cases of suspicious death. In the present case, innumerable widely scattered nodules in both pulmonary fields on MSCT were indicative of miliary tuberculosis (TB). At autopsy, both lungs were submerged in formalin fluid immediately after removal from the body. Miliary TB was finally diagnosed based on microscopic findings. TB is a disease that autopsy room workers need to be aware of to protect themselves. Unfortunately, because little medical information about deceased individuals is usually available before forensic autopsy, the diagnosis of TB is frequently not made until autopsy. This leads to a much higher incidence of TB in autopsy room staff members even if they wear protective clothing. Therefore, MSCT before forensic autopsy may identify suspected cases of miliary TB in advance and thus help to prevent TB infection in forensic autopsy personnel.

Radiological analysis of a naturally mummified body.

A body in an advanced stage of mummification was found in a concrete apartment in Japan. Natural complete mummification is very rare in Japan's humid and temperate climate. We performed multi-slice computed tomography (MSCT) to obtain information on the body prior to autopsy. MSCT clearly illustrated old fractures in the right lower leg. Bone resists destruction during body decomposition. If antemortem medical records of the deceased are available, MSCT scanning can provide information for positive identification. At autopsy, the cervical tissues presented as a dried mass, and it was difficult to separately remove the hyoid bone and thyroid cartilage. Fractures of the large horn of the hyoid bone and superior horn of the thyroid cartilage, which are not observed in all strangulation cases, strongly suggest criminal activity. The diagnosis of these fractures is of great value. In our case, MSCT revealed that there was no fracture in the hyoid bone or ossified area of the thyroid cartilage. Hard tissues are usually well preserved in mummies. Although MSCT images have limits in mummies because of the severe dryness of soft tissues and organs, they could become a useful tool not only for personal identification, but also for the identification of neck compression.

Simultaneous determination of 11 aconitum alkaloids in human serum and urine using liquid chromatography-tandem mass spectrometry.

A liquid chromatography-tandem mass spectrometry method was developed and validated for simultaneous determination of four aconitines (aconitine, mesaconitine, hypaconitine, jesaconitine) and seven of their hydrolysis products (benzoylmesaconine, benzoylhypaconine, 14-O-anisoylaconine, benzoylaconine, aconine, mesaconine, hypaconine) in human serum and urine samples. Extraction was undertaken using a mixed-mode cation-exchange cartridge (OASIS MCX), and D(5)-aconitine was used as an internal standard. Separation of aconitum alkaloids was carried out using an L-column ODS with the mobile phase consisting of 10mM ammonium formate and methanol. The intra- and inter-day precisions were 0.3% to 9.9% and 3.2% to 12.8%, respectively. Intra- and inter-day accuracies were -14.1% to 7.3%, and -10.6% to 8.3%, respectively. The limit of detection and limit of quantification of analytes were 0.04-0.38 ng/mL and 0.12-1.15 ng/mL respectively. This method was applied in an autopsy case and successfully detected aconitines and their metabolites as well as some anti-psychiatric drugs.

Unexpected infant death due to hypoplastic left heart syndrome: a case report.

A female infant was found unresponsive at home. The mother alleged that she delivered the baby at home 13 days prior to the death. The mother did not have any prenatal examinations during the pregnancy and the infant was not examined by a doctor until death. The autopsy revealed that the cause of death was hypoplastic left heart syndrome (HLHS) and the infant's chest showed bilateral breast enlargement. Forensic pathologists may encounter very rare pathological findings with unexpected infant deaths. Some, like HLHS, are serious congenital heart defects related to the cause of death, and others are unique phenomena unrelated to the cause of death such as breast swelling and discharge called "witch's milk." In this case, we observed both findings.