Kotaro Matoba

Unusual injuries on the right hand and forearm caused by unidentified wild animals

An old man was found dead in a rice paddy with his face down in the water. His right forearm and hand were severely injured and the shapes of injuries were unusual. It was initially suspected that the injuries had been caused by a cultivator placed at the site. However, they proved to be postmortem injuries because vital reactions were not observed. The skin was widely torn away. Some edges of the injuries looked like a bite mark and other parts looked like scratches. There were many parallel injuries on the right forearm and hand and footmark-like injuries on the right hand. They were probably caused by wild animals. Judging from the sizes and shapes of the footprint, bite marks and scratches, we estimated that the animal which caused the injuries was weasels.

Application of electrolyte analysis of pleural effusion to diagnosis of drowning.

We measured concentrations of sodium (Na), chloride (Cl), calcium (Ca), and magnesium (Mg) in pleural effusion from forensic autopsy cases to examine whether they were useful for a diagnosis of drowning. We analyzed a total of 51 cases (15 seawater drowning, 10 freshwater drowning, and 26 non-drowning), and determined the following reference values. If the concentration of Na or Cl is under 65 mEq/l, a diagnosis of freshwater drowning can be made. If the concentration of Na is higher than 175 mEq/l, or that of Cl is higher than 155 mEq/l, or that of Ca is higher than 16 mg/dl, or that of Mg is higher than 15 mg/dl, a diagnosis of seawater drowning can be made. We recommend that pleural effusion from the left and the right thoracic cavities should be collected and analyzed separately because large differences may be observed between each side in the case of drowning. If one side corresponds to the reference value for seawater or freshwater drowning and the other side does not, a diagnosis of drowning can still be made according to the obtained value.

Diagnosis of drowning: Electrolytes and total protein in sphenoid sinus liquid

In this study, electrolyte (sodium (Na), chlorine (Cl), and magnesium (Mg)) and total protein (TP) concentrations and volume of liquid in the sphenoid sinus were examined to determine their usefulness to elucidate whether drowning occurred in freshwater or seawater. We examined 68 cases (seawater drowning group: 27 cases, freshwater drowning group: 21 cases, non-drowning group: 20 cases). There was a significant difference in Na, Cl, Mg, and TP concentrations of liquid in the sphenoid sinus among the three groups (seawater drowning, freshwater drowning, and non-drowning groups). To distinguish freshwater drowning from seawater drowning, Na, Cl, and Mg concentrations of liquid in the sphenoid sinus might serve as useful indicators.

Appropriate blood sampling sites for measuring Tg concentrations for forensic diagnosis

Previous studies have reported that thyroglobulin (Tg) concentrations in heart blood are high in cases of asphyxia caused by neck compression such as hanging, strangulation, and throttling and in those with fatal traumatic brain injuries. However, even in cases without these findings presumed to increase the Tg concentration in the previous studies, we previously reported that in some cases the Tg concentration in right heart blood (RHB) and left heart blood (LHB) exceeded the standard value for diagnosis (200 ng/mL) defined in previous studies and the Tg concentration in RHB was significantly higher than that in LHB. In the present study, in our 46 forensic autopsy cases without findings presumed to increase Tg concentration, we separately collected external iliac venous blood (IVB) and external iliac arterial blood (IAB) in addition to RHB and LHB, measured Tg concentrations in RHB, LHB, IVB, and LAB (TRHB, TLHB, TIVB, and TIAB, respectively), and investigated the appropriate blood sampling site for measuring Tg concentrations for forensic diagnosis. TRHB, TLHB, TIVB, and TIAB were 386.3 ± 674.1, 105.8 ± 179.0, 109.2 ± 166.8, and 43.7 ± 90.9 ng/mL, respectively. There were statistically significant differences between TRHB and TLHB, TIVB and TIAB, TRHB and TIVB, and TLHB and TIAB. Tg is more readily diffused by the venous system (RHB, IVB) than by the arterial system (LHB, IAB) because the venous system retains more blood volume after death. Tg is more readily diffused to heart blood (RHB, LHB) than to peripheral blood (IVB, IAB) because of the proximity of the heart to the thyroid gland. Therefore, we conclude that Tg leaks into the vessels around the thyroid gland because of the influences of postmortem changes and subsequently diffuses through the blood after death, and therefore the Tg concentration increases after death. When Tg concentration values are used for forensic diagnosis, it is appropriate to measure them using peripheral arterial blood situated distant from the thyroid gland.

Development for the measurement of serum thiosulfate using LC–MS/MS in forensic diagnosis of H2S poisoning

Thiosulfate measurement is crucial to diagnosis of hydrogen sulfide (H2S) poisoning in forensic toxicology. Although GC-MS method is currently regarded as a standard thiosulfate measurement, it requires complicated sample preparation prior to analysis. This study presents a simple, rapid, and highly sensitive method for the quantitative analysis of serum thiosulfate by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This method is based on selected reaction monitoring and has high sensitivity with a lower quantification limit of 0.5μM. Precision and accuracy of this method meet the basic requirements for quantitative analysis (intra- and inter-day tests have a relative standard deviation of ⩽10.4%; range of analytical recovery is 94.3-102.6%). On the measurements of serum thiosulfate by our developed method, a thiosulfate concentration as 57.5μM was detected clearly in the H2S poisoning case comparing to the non poisoning case in which only a trace amount of thiosulfate was observed.

Unreliability of the use of thyroglobulin concentration in postmortem blood samples in forensic diagnosis

Previous studies have reported that the concentration of thyroglobulin (Tg) in heart blood is high in cases of asphyxia by neck compression such as hanging, strangulation, and throttling and in those with traumatic injuries to the head. However, we have experienced cases in which we observed high Tg concentrations without such findings. Therefore, we analyzed the influence of postmortem changes on Tg concentration. Of 253 forensic autopsies conducted at our institution, we analyzed 44 cases without the findings presumed to increase Tg concentration. We collected right heart blood (RHB) and left heart blood (LHB) separately and measured Tg concentrations in each. The Tg concentration of the RHB in 19 (43%) cases and that of the LHB in 10 (23%) cases was higher than the standard value (200ng/ml) obtained in previous studies. In some cases, we found large differences between the Tg concentrations of RHB and LHB. We suggest that Tg concentration can increase above the standard value and that a difference between the Tg concentration of RHB and LHB arises as a result of postmortem changes. Consequently, if there is a large difference between the Tg concentration of RHB and LHB, the concentration of Tg should not be used as a basis for forensic diagnosis.

Liquid chromatography-tandem mass spectrometry method for the determination of thiosulfate in human blood and urine as an indicator of hydrogen sulfide poisoning

Being a stable metabolite of hydrogen sulfide, thiosulfate has been utilized as an index for hydrogen sulfide poisoning (HSP). Thiosulfate analysis is mainly performed using gas chromatography/mass spectrometry (GC-MS) due to its high sensitivity and specificity. The GC-MS analysis requires two-step derivatizations of thiosulfate, and the derivative is not stable in solution as it has a disulfide moiety. To resolve this stability issue, we developed a novel analytical method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for monitoring the pentafluorobenzyl derivative of thiosulfate (the first reaction product of the GC-MS method) in this study. The established method exhibited high reproducibility despite being a more simplified and rapid procedure compare to the GC-MS method. Phenyl 4-hydroxybenzoate was used as an internal standard because 1,3,5-tribromobenzene which had been used in the GC-MS method was not suitable compound for LC-MS/MS with Electrospray ionization (ESI) negative detection. The linear regression of the peak area ratios versus concentrations was fitted over the concentration ranges of 0.5-250μM and 0.25-250μM in blood and urine, respectively. The validation results satisfied the acceptance criteria for intra- and inter-day accuracy and precision. Blood and urine samples from 12 suspected HSP cases were tested using this method. The thiosulfate concentration detected in the sample coincided well with that determined at the scene of each HSP accident.

Experimental evaluation of freezing preparation for the macroscopic inspection in putrefied brain

PURPOSE To evaluate the usefulness of freezing preparation for macroscopic investigation in advanced putrefied brain. MATERIALS AND METHODS After sealing in individual plastic bags, 10 pig heads were stored at 20°C for 5days allow postmortem change (putrefaction) to progress. After an observation period, they were divided into 2 groups to evaluate the usefulness of the freezing effect in macroscopic investigation. The process over the postmortem period and the freezing process were examined. RESULTS At day-5, the presence of air density was detected between the inner surface of the cranium and the brain parenchyma. Intra-cranial air accumulation presented on CT in all heads. In the control group, the brain parenchyma leaked out from the hole in the meninges, and the gray-white matter difference was clear in 3/72 (4.2%), moderate in 7/72 (9.7%), ambiguous in 17/72 (23.6%), and poor in 45/72 (62.5%). In the freezing group, the brain parenchyma presented homogeneous low density after more than 14h freezing. On opening the cranium, the entire brains were frozen, and the gray-white matter difference was clear in 33/72 (46.0%), moderate in 17/72 (24.0%), ambiguous in 15/72 (21.0%), and poor in 7/72 (10.0%). The freezing group afforded greater clarity in the gray-white matter inspection (p<0.05). CONCLUSION Freezing preparation was useful for the macroscopic investigation of putrefied brain compared with the ordinary autopsy.

Lung weight estimation with postmortem CT in forensic cases

Postmortem computed tomography (CT) is a minimally invasive technique to examine internal organs before a forensic autopsy. The purpose of our study was to estimate lung weight in a forensic setting in cases of various lung states, including fluid accumulation (congestion, edema, hypostasis, and inflammation etc.) using postmortem CT. From January 2016 to July 2018, 111 deceased bodies (62 males and 59 females, aged from 18 to 95 (average 59.6) years) were examined by CT before autopsy. Both lungs of the 111 deceased were analyzed separately, making it a total of 222 samples. We extracted lung fields from CT images manually after semi-automatic detection using an image workstation. The total lung volume and 6 categories of lung volume divided according to their CT density were measured. Multiple regression analysis was performed with lung weight in autopsy as the response variable, while the 6 categories were labelled as explanatory variables. The relation between lung weight in autopsy and lung weight estimated using postmortem CT showed a high Pearsons correlation coefficient (R2 = 0.9106). Using postmortem CT, the lung weight can be estimated in forensic settings.

Freezing preparation for macroscopic forensic investigation in putrefied brain

PURPOSE To evaluate the usefulness of the applied freezing technique in putrefied brain for macroscopic investigation. MATERIALS AND METHODS From October 2015 to September 2016, first the brains of 10 cadavers (control group: male 6, female 4, age 20-80 (mean 61.5), postmortem intervals (PMI) 14-75 (mean 29.7)days) were inspected following the standard practice (without freezing preparation), and then with 10 cadavers (freezing group: male 7, female 3, age 41-88 (mean 60.4), PMI 7-75 (mean 29.2)days) the freezing technique was used before the autopsy. The cut brain was investigated, and the gray-white matter difference was evaluated macroscopically. RESULTS In the control group, the brain parenchyma leaked out like sludge in 5, and there was difficulty maintaining its structure in 7. The gray-white matter difference was well visible in 3, but hard to distinguish in 3, and the total scores ranged from 0 to 9 (mean 4.4) points. In the freezing group, the entire putrefied brain was extracted as a solid organ, the gray-white matter differences were well visible, and the total scores were 6.7-9 (8.3) points. The gray-white matter difference was preserved in the freezing group (p<0.05). CONCLUSION The freezing procedures to evaluate the putrefied brain have been successfully applied, and it could be statistically more useful in putrefied brain investigation than the ordinary procedure. Postmortem CT can be useful to evaluate not only the degree of brain putrefaction, but also the degree of brain parenchyma freezing.