Yuki Ohtsu

Postmortem cytokine levels and severity of traumatic injuries.

The relationship between postmortem serum cytokine levels and severity of traumatic injuries was studied. The postmortem serum levels of interleukin-6 (IL-6) and interleukin-8 (IL-8) of 131 victims who died from traumatic injury were measured by an enzyme-linked immunosorbent assay method and compared with scores of total abbreviated injury scale (total AIS) and injury severity score (ISS) calculated from detailed autopsy reports. A significant positive correlation was observed between IL-6 and total AIS (rs=0.4508, p<0.0001), between IL-6 and ISS (rs=0.3337, p<0.0001), between IL-8 and total AIS (rs=0.6593, p<0.0001), and between IL-8 and ISS (rs=0.5305, p<0.0001). The significant correlation between cytokine levels and anatomical traumatic severity indicated that the cytokine levels are useful objective indexes of traumatic severity. In addition, the total AIS is a suitable marker to evaluate traumatic severity as the coefficient of correlation between the cytokine levels and the total AIS was higher than that for the ISS values.

Japanese monozygotic twins with familial amyloidotic polyneuropathy (FAP) (ATTR VaBOMet)

Twenty-nine-year-old twin brothers having the amyloidogenic transthyretin (ATTR) VaBOMet gene developed the clinical symptoms of familial amyloidotic polyneuropathy (FAP) in 1995. The twins had the same educational background and lived in the same district. FAP manifestations were similar in both cases, although electromyographic examinations revealed sensorimotor polyneuropathy in No. 1 and sensory polyneuropathy in No. 2. DNA analysis revealed that they were monozygotic twins. In addition to environmental factors, genetic factors may play an important role in determining the onset of FAP.

Immunohistochemical demonstration of the distribution of chloroquine (CQ) and its metabolites in CQ-poisoned mice

Chloroquine (CQ) distribution in tissues of acutely poisoned mice was demonstrated by immunohistochemistry using anti-CQ polyclonal antibodies (PAC). PAC recognized 4-amino-7-chloro-quinoline structure and sufficiently reacted with CQ and CQ’s metabolite bisdesethyl-chloroquine. In the brain, CQ and its metabolites (CQs) localized in the region of the choroids plexus, indicating an important role in the blood–cerebrospinal barrier system. In the heart, most regions showed diffused positive staining, and relatively strong reaction was observed in Purkinje cells, indicating an important role in acute CQ toxicity. In the lungs, CQs were observed in the bronchial epithelium, type II pneumocytes, and on the surface of alveolar walls. It was suggested that CQs were excreted to the alveolar wall with surfactant phospholipids, which are produced by type II pneumocytes. In the liver, CQs were concentrated in the centrolobular area rather than in the periportal area, in agreement with CQ’s metabolic pathway. In the kidneys, tubular cells were strongly stained compared to glomerular capsules, and the distal part of renal tubules was better stained than the proximal tubules. These findings suggested that CQs were predominantly excreted or reabsorbed through the distal tubules and the collecting duct. Distribution of CQs in tissues presented here were mostly consistent with the physico-chemical properties of CQ and its metabolites. However, the elucidation of CQs’ localization in Purkinje cells remains open. Further experimental studies at the level of microorganella will be needed to clarify the present result.

Unusual homicide by air gun with pellet embolisation

A homicidal case by an air gun involving a 73-year-old woman who was found dead in her home is described. At autopsy, two gunshot wounds were observed on the left anterior chest and the right back. There was neither exit wound nor powder burns. The fatal pellet, 0.9 g and 5.5mm diameter, penetrated the left anterior chest and the heart to cause cardiac tamponade (200 ml) before lodging in the left subclavian artery. Death was due to cardiac tamponade. This case has exhibited two rare phenomena: homicide by an air gun and pellet embolisation. This is the first homicide case by use of air gun in Japan and demonstrates that although it may be rare, air guns can be used as potential homicidal weapons. This experience will alert our society and others on the potential hazards of air guns we have tended to regard as toys.

Quantitative evaluation of volatile hydrocarbons in post-mortem blood in forensic autopsy cases of fire-related deaths

Volatile hydrocarbons in post-mortem blood from victims of fires were analyzed quantitatively by headspace gas chromatography mass spectrometry. The benzene and styrene concentrations in the blood were positively correlated with the carboxyhemoglobin (CO-Hb) concentration, which is evidence that the deceased inhaled the hydrocarbons and carbon monoxide simultaneously. By contrast, the concentrations of toluene and CO-Hb in the blood were not significantly correlated. This lack of correlation could be explained by two different sources of toluene, with low blood concentrations of toluene arising when the deceased inhaled smoke and high blood concentrations of toluene arising when the deceased inhaled petroleum vapor or other unknown vapors. The quantity of soot deposited in the respiratory tract was classified into four grades (-, 1+, 2+, 3+). The mean CO-Hb concentration in the 1+ soot group was significantly lower than those in the 2+ (p<0.05) and 3+ (p<0.01) soot groups. The blood CO-Hb concentrations in the 1+ soot group were all below 30%. Those indicated that the deceased aspirated smoke that contained both soot and carbon monoxide. The wide variation in CO-Hb concentrations for each soot classification could be caused by the different types of smoke produced by different materials. For example, petroleum combustion with a limited supply of oxygen, like in a compartment fire, may produce a large volume of dense black smoke that contains a large quantity of soot. Soot deposits in the airways and the blood CO-Hb concentration are basic and essential autopsy findings that are used to investigate fire-related deaths. The quantitative GC-MS analysis of blood volatile hydrocarbons can provide additional useful information on the cause of the fire and the circumstances surrounding the death. In combination, these three findings are useful for the reconstruction of cases.

Sudden death of a young woman due to aortic dissection caused by Turner's syndrome

A 24‐year‐old woman was found dead in her bed. There had been an episode of fainting with cervicodynia 1 day before death but no significant past medical history, except for menstrual irregularities. Post‐mortem examination revealed that death was due to hemopericardium caused by rupture of the ascending aorta by thoracic aortic dissection (Stanford type A). Microscopically, weakness of the aorta was due to cystic medial necrosis. On external examination, short stature, a short neck and multiple pigmented nevi were observed, while internal examination revealed coarctation of the aorta and funicular ovaries. Examination of the X chromatin showed a decrease in numbers of Barr bodies in the tissues, and a 45,X/46,XX mosaicism was suspected. It is concluded that the cause of death was aortic dissection due to Turners syndrome.

Death as a result of heat stroke in a vehicle: an adult case in winter confirmed with reconstruction and animal experiments

A 54-year-old man was found dead in the drivers seat of his vehicle on a winters day. Investigations of the vehicle revealed that the engine was running, and the car heater was left on with the maximum temperature and velocity. The body was found excessively sweating. Rectal temperature of the body was 43 degrees C at 10 h post mortem. In autopsy, several superficial skin burns were observed on the face, the shoulders and the legs. The lungs were heavily congested and hemorrhagic. The liver showed typical alcohol-induced micronodular cirrhosis. The alcohol concentrations were 0.17% in the blood of both the left and the right heart, 0.17% in the femoral-vein blood, 0.21% in the bladder urine and 0.34% in the gastric contents. A reconstruction experiment demonstrated that the temperature inside the vehicle rose rapidly and reached 50-58 degrees C in 3 h. Animal experiments showed that the temperature threshold for rats to succumb to heat was between 40 and 45 degrees C. This case shows that heat stroke in a vehicle can occur in adults with chronic diseases or alcoholism, such as in this particular case, even in the winter.

Single-chain variable fragment technology in forensic toxicological analysis: production of an antibody to fluvoxamine

Immunoassay techniques are widely used for drug screening in the fields of forensic toxicology and emergency medicine, because of their simple procedures and rapid outcome of results [1, 2]. To create an immunoassay method for a compound, the most laborious step is the production of an antibody that is specific to the compound. Here, we present a new recombinant antibody technology for producing a single-chain variable fragment (scFv). scFv is a small antibody molecule that retains high antigen specificity and binding activity of the original whole immunoglobulin [3]. One advantage of scFv is its simple production using bacterial cell cultures, which also provide variable functionalities by introducing mutations. Furthermore, creating scFv phage libraries, which display various scFvs, might be useful in searching for drug-reactive elements. This seems to be a powerful tool for rapid creation of new antidrug antibodies without immunizing animals. Recently, the frequency of psychotropic drug poisoning has increased in Japan [4]. Among such drugs, fluvoxamine (FLV), one of the selective serotonin reuptake inhibitor (SSRI) antidepressant drugs, is widely used for treatment of depression and obsessive compulsive disorders [5]. Although SSRIs are relatively safe as compared with conventional antidepressant drugs, such as tricyclic antidepressants and monoamine oxidase inhibitors [6], the number of FLV poisoning cases has increased according to the increase in its prescription [7, 8]. In this study, we created an anti-FLV scFv using novel recombinant antibody technology. FLV malate was obtained from Sigma-Aldrich (St. Louis, MO, USA). Mercaptosuccinyl bovine serum albumin (MS-BSA) was a generous gift from Prof. K. Fujiwara, Sojo University (Kumamoto, Japan). All other solvents and chemicals were of analytical grade, and purchased through local suppliers. BALB/c mice (female, 4 weeks old; Kyudo, Kumamoto, Japan) were maintained in the Center for Animal Resources and Development, Kumamoto University, Kumamoto, Japan, and were kept in an environmentally controlled room (22 ± 2 C, 50–70 % humidity, illuminated from 0700 to 1900 hours). All procedures were approved by the Kumamoto University Ethics Review Committee for Animal Experimentation. The immunogen (BSA–FLV) for induction of anti-FLV antibodies was prepared as described previously with a slight modification [9]. Briefly, FLV maleate (4.3 mg; approximately 10 lmol) in 1.0 ml of 50 mM phosphate buffer (pH 7.0) was mixed with N-[c-maleimidobutyryloxy] succinimide (GMBS) (0.25 mg; approximately 0.89 lmol), and incubated at room temperature with stirring. MS-BSA, estimated to contain 18 thiol groups per BSA molecule, was diluted with 3.0 ml of 0.1 M phosphate buffer (pH 7.0), and added immediately to the This article is for the special issue TIAFT2012 edited by Osamu Suzuki.

Liquid chromatography-tandem mass spectrometry detection of benzalkonium chloride (BZK) in a forensic autopsy case with survival for 18 days post BZK ingestion

We report a forensic autopsy case of an elderly man who ingested unknown amount of germicidal disinfectant containing 50% benzalkonium chloride (BZK). He survived for 18 days after BZK ingestion and then died because of pneumonia. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used to detect three BZK compounds (C12-BZK, C14-BZK and C16-BZK) in the blood. Extraction of BZK was carried out according to a modified QuEChERS method. Chromatographic separation was achieved on an ODS column and detection was performed in selected reaction monitoring mode. The accuracy and the precision were acceptable for quantitative analysis in the concentration range of 10-200 ng/mL for the three BZK compounds. BZK was detected in heart and femoral vein blood samples even 18 days after BZK ingestion. Taking into consideration clinical information during 18 days hospitalization and the autopsy findings, the cause of death was attributed to BZK poisoning. Several toxico-kinetic factors regarding absorption and excretion of BZK in the body were also discussed to elucidate the detection of BZK such a long time after ingestion.

A simple dot enzyme-linked immunosorbent assay for ABO blood typing of biological fluid and stains: effects of heating samples

A simple dot enzyme-linked immunosorbent assay (Dot-ELISA) using commercially available monoclonal anti-A and anti-B antibodies and biotinylated anti-H lectin was developed for ABO blood typing of biological fluid and stains. Its application to forensic practice was examined with 117 saliva samples and their stains, and practical case samples of 8 seminal, 6 vaginal and 45 aged salivary stains. In the simple Dot-ELISA, a new step to heat biological samples was introduced in the system in order to block unfavorable non-specific reactions of the samples with secondary enzyme conjugate. The simple Dot-ELISA could determine accurately the ABO blood type of a small amount of secretors and non-secretors salivary samples. In practical tests of seminal, vaginal and salivary stains, all results were confirmed to be identical to those determined by the conventional absorption-inhibition test and the absorption-elution test. The simple Dot-ELISA is considered to be accurate, rapid, simple, sensitive and easy to perform in routine forensic practice. It is also a unique and helpful method to determine the ABO blood types of various biological samples.