Detection of diatom in formalin-fixed tissue by proteinase K digestion

The diatoms detection has been proposed to be useful in the diagnosis of drowning. Enzymatic digestion of unfixed lung tissues and other organs with proteinase K is widely employed to detect diatoms. Handling unfixed organs or blood from the bodies with some infectious diseases could prove to be dangerous. In this study, we examined the application of enzymatic digestion for diatom detection to formalin-fixed lung obtained at autopsy. Furthermore, we assessed the effect of hydrogen peroxide on the contamination of the lung specimen with foreign bodies inhaled in the course of drowning, smoking, or air pollution. Formalin-fixed lung was heated in 0.01 M Tris–HCl buffer (pH 7.5) containing sodium dodecyl sulfate (SDS) (tissue lysis-buffer), with or without glycine. Thereafter, the lung was subjected to enzymatic digestion with proteinase K. A part of formalin-fixed or unfixed samples digested with proteinase K were incubated with hydrogen peroxide at 80 °C for 6 h or 12 h, while the residues were processed without incubation. Formalin-fixed samples heated in tissue lysis-buffer with glycine could be digested with proteinase K; further, the number and proportion of diatoms detected in both formalin-fixed and unfixed samples were observed to be similar. The results suggest that enzymatic detection of diatoms can be applied to formalin-fixed organs by heating the samples in glycine-containing tissue lysis-buffer. As the use of formalin-fixed tissue for diatom detection can decrease risk of contamination by pathogenic organisms during the course of enzymatic digestion, the method presented in this study would be beneficial, to some extent, to individuals performing diatom analysis. Moreover, our results suggest that archival organs stored in formalin solution could be available in diatom detection over a long time-period following autopsies. Clearer image of diatoms was observed in the specimen incubated with hydrogen peroxide for 6 h, in which inhaled foreign bodies were discolored, than those not subjected to incubation. Therefore, incubation of sample digested with hydrogen peroxide in the limited time would be helpful for quantitative and qualitative diatom analysis.     

A transnasal intracranial stab wound by a plastic-covered umbrella tip

A 48-year-old man died from a transnasal intracranial stab wound caused by an umbrella. The track of the stab passed from the right nostril, through the sphenoid sinus, the left side of the sella turcica and anterior clinoid process, and finally reached the surface of the brain. The stab wound crossed the left internal carotid artery, causing an exsanguination and aspiration of blood into the airway, resulting in death. It is extremely rare that an umbrella tip used during a struggle would stab the nostril of the victim. Transnasal intracranial stab wounds can be overlooked and require sensitive handling.     

Blunt pancreatic trauma by a wheelchair user restraint system during a traffic accident.

Traffic accidents are the most common cause of blunt pancreatic trauma, and most injuries occur in unrestrained drivers. Blunt pancreatic trauma in a passenger or a restrained driver is rare. In this report, we describe a case of blunt pancreatic trauma caused by a restraint system for wheelchair users during a traffic accident.     

Autopsy and genetic diagnosis of 21-hydroxylase deficiency with bilateral testicular tumors in a case under no medication for over one year

The autopsy findings of an adult patient with 21-hydroxylase deficiency are presented. Genetic analysis of the 21-hydroxylase gene (CYP21A2) was performed for accurate diagnosis of congenital adrenal hyperplasia (CAH), and bilateral testicular tumors were characterized. We report a 29-year-old Japanese man who was diagnosed with CAH (21-hydroxylase deficiency) in infancy and had continued steroid therapy until the age of 28. However, for more than one year, he had not been treated for CAH and was found dead. In the medico-legal autopsy findings, both adrenal glands were enlarged, and hypertrophy of adrenal cortices and bilateral testicular tumors positive for melan-A were observed. Genomic DNA was prepared from cervical lymph nodes collected during autopsy, and CYP21A2 was PCR amplified and sequenced directly using newly designed primers. From the morphological findings, the bilateral testicular tumors were considered to be adrenogenital syndrome (TTAGS). Through the whole sequence of CYP21A2, the intron 2 splice mutation (656)A to (656)G was found. TTAGS were thought to be adrenal rests enlarged by ACTH stimulus. From the autopsy findings and the result of genetic analysis, he was diagnosed with the salt-wasting form of 21-hydroxylase deficiency and his cause of death was presumed to be heart failure based on abnormal electrolytes.     

Identification of male bloodstains by dot hybridization of human Y chromosome-specific deoxyribonucleic acid (DNA) probe

The sex determination of bloodstains was performed using a human Y chromosome-specific (DNA) fragment of 1.9-kb length as a hybridization probe. The DNA samples were taken from 1- and 4-week-old bloodstains of males and females, respectively. Strong signals with male DNA were observed by Y-probe, while faint signals with female DNA were detected. In addition, clear signals were observed in the extract samples from male bloodstains (16-week-old) on paper. Dot hybridization of the Y-probe would be widely applicable to studies on sex determination of medicolegal materials such as blood, bloodstains, teeth, and cadaverous parts.     

Reflections on the Kyoto conference on global warming

The Third Conference of Parties to the United Nations Framework Convention on Climate Change, was held in Kyoto in December 1997. It ended successfully with the adoption of the Kyoto Protocol, which lays down internationally binding provisions for handling the problem of global warming in the twenty‐first century, but the negotiation process was extraordinarily difficult, due to the complex nature of the issue of global warming, and to the fact that the national interests of every nation were closely involved. As Japan's ambassador for global environmental affairs, Toshiaki Tanabe was chief negotiator for Japan, and gives here an insider's view of how that successful outcome was reached. The requirement for Japan to reduce greenhouse gas emissions by 6 percent is a heavy burden, he says, and poses a serious challenge.     

Longitudinal evaluation of immunohistochemical findings of milk aspiration: an experimental study using a murine model

To examine the longitudinal change of pathological findings of the lung and other organs in milk aspiration, an experimental study using a murine model was carried out. Either 0.5 or 1.0 ml cow's milk was instilled into the trachea of rats. From immediately after to 14 days after instillation, the animals were sacrificed, and the lungs, liver, kidneys, and spleen were removed. The results of immunostaining with anti-human α lactalbumin antibody indicated that not only the lung but also the kidney and spleen showed a positive reaction against the antibody over time. Experimentally aspirated milk was detectable in alveoli until 2 days after instillation. It was also detectable in renal tubules from 1 to 6h after instillation. Macrophages containing granules of aspirated milk were observed in splenic red pulp from 3h to 14 days after instillation. Detection of aspirated milk in other organs except the lung would be clear evidence of intravital milk aspiration and would suggest previous or recurrent milk aspiration.     

Isoelectrofocusing in the study of the BfF system: existence of two common subtypes of the BfF allele in a Japanese population

The Bf gene frequencies including BfF' allele and BfF' allele in a Japanese population were studied by using the PAGIF method. The results showed the Bf gene frequencies: BfF' allele = 0.0778, BfF' allele = 0.1007 and BfS allele = 0.8215.