Establishment of a random forest regression model to estimate the age of bloodstains based on temporal colorimetric analysis.

Bloodstain age estimation is important in forensic science. Although several studies have used spectroscopy to estimate bloodstain ages, this method has not yet been practically applied due to the need for expensive equipment and low reproducibility. Thus, we aimed to develop a bloodstain age estimation model that can be easily performed using a spectrophotometric colorimeter. First, bloodstains were prepared by placing blood obtained from five healthy volunteers on a plastic plate. The bloodstains were kept on conditions with various brightness and temperatures. Then, each bloodstain was dissolved in saline every 24 h to a final concentration of 1%, measured with a spectrophotometric colorimeter, and subjected to machine learning to generate a random forest regression (RFR) model, and finally, the prediction accuracy of the bloodstain age was verified. We also elucidated the mechanism of the color changes utilizing aminoguanidine, which is an inhibitor of Maillard reaction. Finally, we measured the time-dependent color changes of the blood fluids obtained from healthy volunteers and examined if the method could be potentially applied to estimate postmortem interval (PMI). Our results showed that the RFR model estimated the bloodstain age with no substantial assessment, and it was applicable to bloodstains, regardless of the brightness or temperature. The color changes were affected by the addition of aminoguanidine. Furthermore, the method could be applied to blood fluids, suggesting its potential usefulness for PMI estimation. Considering its feasibility, the present method could potentially be introduced to practical forensic sciences in the near future.

Postmortem genetic analysis of 17 sudden cardiac deaths identified nonsense and frameshift variants in two cases of arrhythmogenic cardiomyopathy.

Sudden death, or unexpected natural death of a healthy individual, is a serious problem in all nations. Sudden cardiac death (SCD) mainly due to ischemic heart diseases is the top cause of sudden death. However, there are pathophysiological conditions, referred to as sudden arrhythmic death syndrome, in which no apparent lesion can be identified even after complete conventional or ordinary autopsy. While postmortem genetic analyses have accumulated evidence about underlying genetic abnormality in such cases, the precise relationships between genetic background and the phenotype have been largely elusive. In this study, a retrospective investigation of 17 autopsy cases in which lethal arrhythmia was suspected to be the cause of death was carried out. Genetic analysis focusing on 72 genes reported to be associated with cardiac dysfunctions was performed, in combination with detailed histopathological and postmortem imaging examination, and a family study. As a result, in two cases of suspected arrhythmogenic cardiomyopathy (ACM), we found a nonsense variant in PKP2 and frameshift variant in TRPM4 gene. In contrast, the other 15 cases showed no morphological changes in the heart despite the presence of a frameshift variant and several missense variants, leaving the clinical significance of these variants obscure. The findings of the present study suggest that nonsense and frameshift variants could be involved in the morphological abnormality in cases of SCD due to ACM, while missense variants alone rarely contribute to massive structural changes in the heart.

Development of a detector tube for screening tadalafil and its analogues in adulterated sexual enhancement products.

Sexual enhancement products adulterated with phosphodiesterase 5 inhibitors (PDE-5i) pose a serious public health concern. Tadalafil and its analogues (Tds) are PDE-5i frequently detected as adulterants. In this study, a Td detector tube for the rapid detection of Tds was developed based on the color change reaction between sulfuric acid and Tds. The specificity of this test method was evaluated using 13 Tds, all of which elicited positive results. Additionally, 30 commonly found adulterants in dietary supplements, 11 active pharmaceutical ingredients of psychotropic drugs and 18 food ingredients were tested and obtained no false-positive results, except levomepromazine. The test tube accurately detected the presence or absence of Tds in 54 commercially available products. The visual detection limit was 2-50 and 5-20 µg/ml for Tds and tadalafil-spiked samples with matrix, respectively. The applicability of the developed detector tube to a semiquantitative test using digital image analyses were investigated using red, green, and blue color values. The results of the recovery test suggested that the tube test was affected by the dark-colored matrix. The results of semiquantitative analyses of tadalafil for five marketed products were consistent with the liquid chromatographic quantification results, except for the blue value. The detector tube developed in this study can facilitate with the rapid screening of Tds in adulterated sexual enhancement products.

[Identification of Descarbonsildenafil in an Adulterated Dietary Supplement and Evaluation of Its Inhibitory Activity for Phosphodiesterase Type 5 (PDE5)].

Some illegal dietary supplements contain phosphodiesterase type 5 (PDE5) inhibitors, such as sildenafil, for exerting "therapeutic" effects in erectile dysfunction. This is apparently dangerous, and thus, should be appropriately regulated. Identification of descarbonsildenafil was first reported in Singapore in a coffee sample labeled to exert male sexual performance enhancement effects. However, it is unclear whether the compound possesses PDE5 inhibitory activity. We encountered during our survey of dietary supplements, a sexual enhancement product commercially available in Tokyo, in which a peak presumed to be of descarbonsildenafil was detected by LC-UV and electrospray ionization-tandem MS (ESI-MS/MS). The compound was isolated and identified as descarbonsildenafil with liquid chromatography-quadrupole time-of-flight-mass spectrometry (LC-QTOF-MS), NMR, and X-ray crystal structural analysis. In addition, descarbonsildenafil showed PDE5 inhibitory activity in PDE5 inhibition assay, and its IC50 value for PDE5A1 was found to be 30 nmol/L. The results of INADEQUATE NMR and X-ray crystal structural analysis in this study provide information for the identification of descarbonsildenafil. Since this study indicates that this compound is a PDE5 inhibitor having adequate activity, it is regulated as a drug component in Japan.

Severe Cardiac Rupture by Only One Blow to the Chest in a Young Boy: An Autopsy Case.

Cardiac rupture by blunt chest trauma is commonly seen after motor vehicle accidents and falls; however, it is rarely caused by a blow to the chest. We herein report an autopsy case of a high school boy who sustained severe right ventricular rupture by only one knee kick to the chest during a quarrel. He was hospitalized and developed cardiopulmonary arrest. Emergency surgery was performed, but the patient died. The autopsy revealed no external severe trauma or deformation, but the side wall of the right ventricle contained a large V-shaped laceration. The other thoracic organs had no injuries. This case illustrates that severe cardiac rupture can occur by only one blow to the chest. Blunt cardiac injuries can occur even if no severe injuries are present on the body surface. We should consider the possibility of severe cardiac injuries regardless of the presence of external injuries.

Semiquantitative screening of trace combustion-derived volatile substances in the blood of fire victims using NeedlEx® headspace gas chromatography/mass spectrometry.

BACKGROUND: Various autopsy findings are used for the diagnosis of burning death, including inhalation injury, soot in the respiratory tract, vital reaction, blood carboxyhemoglobin (COHb), and blood cyanide. However, not all findings are necessarily complete, and autopsy diagnosis can be difficult in cases where there are few findings. In this study, we analyzed combustion-derived volatile substances (CVSs) in post-mortem blood in fire-related cases, focusing on those that occur when buildings and furniture thermally decompose during a fire, and evaluated these as potential new autopsy findings. METHODS: The blood CVSs of 61 fire-related cases from 2013 to 2015 were analyzed by a rapid and operationally simple method that employed a gas chromatograph-mass spectrometer coupled to a NeedlEx®, an extraction needle packed with a medium that selectively adsorbs volatile organic compounds. RESULTS: A total of 39 types of aliphatic and aromatic hydrocarbons were detected in the blood of fire victims. They could be detected even in post-mortem blood with COHb <10%, cyanide concentration <0.25µg/ml, and no soot in the respiratory tract. Styrene was detected in all cases except for one case of burning suicide with kerosene. The blood CVSs could be readily differentiated between the victims of building fires, self-immolation with kerosene, and self-immolation with gasoline: the presence of alpha-methylstyrene with a large amount styrene indicated a building fire; the detection of C9-C12 aliphatic hydrocarbons and C3 alkylbenzenes suggested a kerosene-related fire; and the detection of large amount of toluene and C3 alkylbenzenes indicated a gasoline-related fire. The number of blood CVSs detected for the building fire victims showed correlations with COHb level, cyanide concentration, and the amount of soot. With self-immolation, the number of blood CVSs detected showed weak correlation with COHb level, cyanide concentration, or amount of soot. CONCLUSION: Analysis of blood CVSs enabled the identification of the type of fire-related death, which is impossible by conventional autopsy findings.

[Suicide case of carbon dioxide poisoning using dry ice].

We had a suicide case suspected to result in death from carbon dioxide poisoning by dry ice in the car. A clay cooking stove with charcoal was in the car, but the charcoal had no burning sign. CO hemoglobin saturation degree of the suicide victim's blood was 0%. Moreover, there were signs that 50 kg of dry ice was brought in the car. To clarify the cause of death, reproducibility testing was carried out by using a car under the same conditions. CO2 concentration in it increased to 22% and O2 concentration decreased to 16% within 20 minutes. From these observations, his death was considered to be caused by hypoxia and CO2 narcosis. CO2 in the suicide victim's blood was higher than those in the blood of healthy persons, and the same range was visible in the blood of fire victims. These data might support above supposition concerned with the cause of death. Blood analysis will be helpful in clearing the cause of death by CO2 poisoning.

[Development of salicylic acid dipstick test].

A dipstick test was successfully devised for the screening of salicylic acid in urine. It, named "salicylic acid dipstick test", is TLC plastic sheet which was soaked into 5% (w/v) of ferric chloride aqueous solution, then was covered with Oblate and dried. The test paper was dipped into the sample, then it immediately turned purple under the condition of more than 100 microg/ml of salicylic acid in urine. This device was useful for the emergency screening of salicylic acid in acute poisoning cases with aspirin.

[Application of extraction needle using selective and enhanced polymer in the analysis of samples containing poisonous material].

Low concentrations of gas components usually need to be condensed before analyzing them by gas chromatography (GC). Extraction Needle using Selective and Enhanced Polymer (EN-SEP) is a recently developed devise for condensing the components that are difficult to detect by using GC. EN-SEP consists of a needle which contains methacrylate co-polymer. Gas components are adsorbed and condensed on the polymer by passing through the needle. This paper describes the usefulness of EN-SEP in a criminal case. EN-SEP was successfully applied to condense and detect a trace of propylene glycol monomethyl ether contained in a soft drink by using gas chromatography/mass spectrometry.

[Development of salicylic acid detector tube].

A detector tube was successfully devised for the screening of salicylic acid in urine. It, named "salicylic acid detector tube", consists of glass tube in which silica gel coated with 5% (w/w) of ferric chloride is enclosed. A pipette rubber cap was attached to an end of the tube, and another end was inserted into urine sample. The sample was then introduced into the tube, the color of the reagent immediately turned purple under the condition of more than 50 microg/ml of salicylic acid in urine. This device was useful for the emergency screening of salicylic acid in acute poisoning cases with aspirin.

Analysis of toluene in exhaled air of glue sniffers and workers using thinner at work place.

Toluene concentrations in exhaled air of workers using thinner at work place and glue sniffers were measured by gas chromatography (GC). Toluene concentrations in exhaled air of workers were significantly lower than those of glue sniffers. This suggests that a worker does not inhale as much thinner as a sniffer and that the analysis of thinner in exhaled air can be an indicator for its inhalation. It is also considered that the analysis of toluene concentration in the exhaled air gives an important clue whether the person inhaled thinner during working or self-abuse.

[A case of chloropicrin detection by purge and trap gas chromatography/mass spectrometry].

Chloropicrin (CP) is an agricultural chemical used as insecticide or fungicide, which occasionally causes accidental poisoning and may used in criminal cases including suicide and homicide. Although CP is usually analyzed using gas chromatography/electron capture detection technique (GC/ECD), further identification by gas chromatography/mass spectrometry (GC/MS) may be required from the view point of forensic chemistry and also clinical toxicology. However, it is very difficult to detect a trace of CP by routine GC/MS. The present paper describes a criminal case, in which GC/MS equipped with curie point purge and trap sampler was successfully applied to detect a small amount of CP.