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The number of diatoms detected in the kidneys and liver varies considerably when comparing various reports, but the reason remains unclear. We previously showed that the reuse of Kjeldahl flasks is susceptible to generating false positives; false-positive results were observed in 17 of 60 tested flasks, and a maximum of 290 diatoms were detected. Furthermore, these data suggested that the number of diatoms in kidney and liver tissue may be much lower than previously thought. Therefore, only new Kjeldahl flasks were used to perform diatom tests on 80 autopsy cases (drowning and non-drowning victims) in this study. When combined with the 20 cases in a previous study, a total of 100 cases were analyzed. Four grams of lung, 10-30 g each of kidney and liver, 10 mL of blood, and 50-100 mL of water (discovery site) were examined. A large number of diatoms (n = 400) were found in organs other than the lungs in only 1 of 56 cases of suspected drowning (excluding cases of suspected drowning in baths). Together with the previous 20 cases, this single case represented only 1 of 76 cases (1.3 %). In closed organs and blood samples, 3 diatoms were found in 2 of 56 victims (3.6 %), and 1 or 2 diatoms were found in 8 victims (14 %). However, 5 diatoms were found in 1 of 10 bathtub drowning victims and 12 non-drowning victims (4.5 %), and 1 or 2 diatoms were found in 6 victims (27 %). Moreover, 1 or 2 diatoms were found in negative control flasks in 3 of 80 victims (3.8 %). The results showed that distinguishing false-positive results is difficult when a small number of diatoms are found in the kidneys and liver. Thus, the presence of diatoms in closed organs and blood seems to be unreliable as evidence of death by drowning, at least in the water environments in our region (freshwater areas: 23-26,000 diatoms/mL, median 200 diatoms; brackish water areas: 26-1200 diatoms/mL, median 210 diatoms; seawater area: 2-1000 diatoms/mL, median 24 diatoms). These results also reconfirmed that the reuse of flasks is the most important cause of false positives. Conversely, the presence of a large number of diatoms in lung samples (1-850,000 diatoms/g lung tissue; median (right lung): 440 diatoms, (left lung): 320 diatoms) is very informative and is an effective diagnostic aid in many cases.

Objectives: To compare “black ring-shaped burn” (BRSB) and charring using spectral computed tomography (CT). Methods: Spectral CT was performed using chicken pectoralis minor muscle, processed in three ways and unprocessed as a control: a) BRSB generated by bringing the negative pole surface of a 3 V button battery (BB) into contact with the muscle; b) BRSB caused by a 1.5 V BB; c) charring caused by broiling; and d) control. Attenuation values were compared between BRSB and charring. Muscles were formalin-fixed and stained with Perls’ Prussian blue. Results: Attenuation values from polychromatic 120-kVp images were significantly higher for BRSBs than for charring. In the spectral Hounsfield unit curve, attenuation values for BRSBs were higher for lower energy. Histopathologically, BRSBs stained positively with Perls’ Prussian blue. Conclusions: This study using spectral CT revealed that BRSB contains metal and confirmed the presence of Fe3+ histopathologically. BRSB differs from charring due to burns. Advances in knowledge: The exact composition of BRSB remains unclear, but this report is the first to show that BRSB differs from charring using spectral CT. Clarification of the composition of BRSB is expected to facilitate the development of more effective BRSB removal therapy.

A case of clinically unsuspected fatal interrupted aortic arch (IAA) is described. A 17-day-old Japanese girl unexpectedly entered respiratory arrest at home. On autopsy, the heart was hypertrophic, with no apparent connection between the ascending and descending aortas. The ascending aorta branched into common carotid and right subclavian arteries, whereas the left subclavian artery arose from the descending aorta, which was supplied by the ductus arteriosus, indicating type B IAA. In addition, ventricular septal defect, bicuspid aortic valve, patent foramen ovale, and thymic aplasia were identified. The immediate cause of death was assumed to be "ductal shock." Because of the known strong association between type B and 22q11.2 deletion, her parents received genetic counseling and requested chromosomal analysis of the child. Fluorescence in situ hybridization worked well on a frozen blood sample, identifying the suspected deletion. This case was thus diagnosed as 22q11.2 deletion syndrome exhibiting IAA and thymic defect.

The first quantitative study on the effect of visible light irradiation on the luminol reaction, used forensically, was conducted using whole human blood aqueous solutions (hemolytic state) and an LED lamp. Whole human blood aqueous solutions under an air atmosphere were irradiated with visible light, resulting in the maximum chemiluminescence (CL) intensity (@ 440 nm) increasing about 1.7-fold due to acceleration of the luminol reaction rate. No acceleration effect was observed under an argon (Ar) atmosphere, or under an air atmosphere in the presence of sodium azide (NaN3; a scavenger of singlet oxygen (1O2)). Furthermore, no conversion from Fe(II) hemoglobin to Fe(III) hemoglobin (methemoglobin) was observed in the absorption spectrum following irradiation. We suggest that these effects are due to easier approach of the luminol reagents to heme following damage of the globin protein around the heme, or damage to the red blood cell membrane, induced by 1O2 generated by an excited state of heme.

Diatom analysis is very effective for positive diagnosis of water inhalation in drowning. However, conventional strong acid diatom testing is laborious and potentially dangerous. We propose a simple, fast, and safe protocol using inexpensive reagents such as papain, SDS, and 5 N HCl for extracting diatoms from lung, kidney, and liver tissues. First, we determined optimal conditions for papain digestion using porcine tissues. Papain digestion was clearly superior to Proteinase K digestion. Next, for assessing the assay effectiveness in practical cases, the papain digestion protocol was applied to 80 tissue samples from 20 suspected drowning victims. Left and right lung tissues (1 g each) were digested in 15-mL conical centrifuge tubes. Kidney and liver tissues (10 g each) were extracted in 175-mL conical centrifuge bottles. Papain dissolved all organs sufficiently and permitted clear visualization of diatoms, although papain’s solubilization activity was still inferior to strong acid digestion. The proposed enzymatic method requires only a low-speed centrifuge and water bath. Diatoms typically can be extracted from tissue samples within 3–5 h. The cost of protease is reduced some 6-fold by using papain in place of Proteinase K. Thus, the proposed method can be useful as a less-laborious, less-hazardous, and less-costly minimal test when the conventional strong acid digestion method is not performed due to personnel, equipment, budgetary limitation, or environmental and safety considerations.

A case involving an adult dismembered body with myositis ossificans (MO) is described. A woman in her 20s was found dead in her cohabitants' room. The body had dismembered into 15 pieces. Computed tomography showed ectopic mineralization in the bilateral upper arms and thighs. On autopsy, the skin and soft tissue were in the process of adipocere formation. Darkened areas that appeared to be subcutaneous hemorrhage were distributed on the face, left chest, and back. Bilateral upper arms and thighs showed organized granulations adherent to underlying bone. Microscopically, these organized lesions represented mature bone tissue in the periphery, cartilage tissue in the middle layer, and fibrotic tissue in the central part. These were typical findings of MO. Myositis ossificans and subcutaneous hemorrhage seemed to be evidence of antemortem repetitive physical abuse. In cases of dismemberment, the absence of organs and parts of the body provide limitations to determining cause of death. In forensic fields, adult autopsy cases involving MO are rare. This is an unusual case in which MO revealed antemortem physical abuse despite dismemberment of the body.

Low rates of diatom positivity in the closed organs of drowning victims present challenges for diatom testing. High positivity rates in closed organs of non-drowning victims also raise an important issue. These contradictory findings were common in diatom testing studies undertaken during the 1960-80 s, but the reasons remained unclear. In the present study, we identified one of the most important factors associated with false-positive results in diatom testing using strong acid. One to 290 false-positive diatoms were found in reused Kjeldahl flasks that were thoroughly washed after the first diatom testing and kept free of tissue before the second testing. False-positive results occurred in 11 of 20 cases when more than approximately 10,000 diatoms were present in digested tissue or water samples. Reused flasks were found to contain many common diatoms (30 µm), including Cocconeis, Cymbella, Diatoma, Gomphonema, Navicula, and Nitzschia, in agreement with reports of diatoms identified in closed organs. Surprisingly, such false-positive results occurred even at the sixth diatom testing using the same flasks kept free of tissues in each analysis. In contrast, no diatoms were detected in any reagent or associated with other glassware. Thus, reuse of Kjeldahl flasks can readily cause false-positive results that cannot be completely prevented by cleaning the flasks using alkali detergents, as evidenced by detection of diatoms even after six tests. We assume that diatoms causing false-positive results are partially melted by heating and fixed onto the flask's inner surface glass, as the diatom frustule consists primarily of SiO

We developed a novel molecular tool for assisting the diagnosis of death by drowning and evaluated its validity in forensic practical cases. Two novel sets of loop-mediated isothermal amplification (LAMP) primers were designed to detect either representative freshwater (Aeromonas) or marine (Vibrio, Photobacterium, Listonella) bacterioplankton (aquatic bacteria) in one tube using the LAMP technique. The assay involves only mixing template DNA with seven reagents and incubating at 64 °C for 80 min and does not require special or expensive equipment because detection is based on visual observation under natural light. The assay’s excellent specificity was also demonstrated using 17 standard (control) strains and 124 other bacterial strains cultured from drowning and non-drowning victims in our previous studies. We then assayed 299 specimens (135 lung, 164 blood) from 72 victims, including 45 who had drowned in rivers, ditches, seas, and around estuaries. LAMP assay results could provide effective information to assist the diagnosis of death by drowning in practical cases. The LAMP assay would be useful for suspected drowning cases, as it is a less-laborious and less-expensive minimal test when death by drowning is sufficiently confirmed or negated from only autopsy findings and environmental data or when diatom testing is not performed due to logistic, personnel, or budgetary limitations. Moreover, the assay could serve as a simple additional test when the density of diatoms in the lungs is very low due to low density in the water.

To determine the feasibility of human identification through the two-dimensional (2D) fusion of postmortem computed tomography (PMCT) and antemortem chest radiography. The study population consisted of 15 subjects who had undergone chest radiography studies more than 12 months before death. Fused images in which a chest radiograph was fused with a PMCT image were obtained for those subjects using a workstation, and the minimum distance gaps between corresponding anatomical landmarks (located at soft tissue and bone sites) in the images obtained with the two modalities were calculated. For each fused image, the mean of all these minimum distance gaps was recorded as the mean distance gap (MDG). For each subject, the MDG obtained for the same-subject fused image (i.e., where both of the images that were fused derived from that subject) was compared with the MDGs for different-subject fused images (i.e., where only one of the images that were fused derived from that subject; the other image derived from a different subject) in order to determine whether same-subject fused images can be reliably distinguished from different-subject fused images. The MDGs of the same-subject fused images were found to be significantly smaller than the MDGs of the different-subject fused images (p

We collected 68 fresh, brackish, and seawater samples from various sites around the estuaries of 2 rivers at high and low tides. Seawater flowed approximately 2.4 (salinity, 2.2% at the site) and 1.2 km (1.8%) upstream of the estuaries, but the surface comprised essentially fresh water up to the mouth. Sites contained 69 to 22,200 diatoms/50 mL of water, and the numbers varied by depth and at sites separated by only approximately 1.2 km. Diatoms ranged from 2.8 to 429 μm (mean range, 16.1-59.2 μm) in size. Large pennate diatoms populated fresh water areas, and most sedimented before reaching the sea. Numbers of pennate diatoms of less than 20 μm were decreased in areas of seawater. Numbers of centric diatoms tended to increase nearer the sea, and seawater contained large centric diatoms. Brackish water containing large volumes of seawater was easily discriminated by assemblages of marine diatoms, unlike that containing a little seawater, because marine diatoms could be found in fresh water around estuaries. Tides and the nature of the river often altered diatomaceous assemblages at the same estuarial sites. Caution is recommended for forensic interpretation of aqueous media to deduce drowning sites.

Numbers and types of bacterioplankton proliferating in blood samples mixed with water of various salinity levels were examined to determine the characteristics of species associated with salinity. Water samples (total n=88) were collected from the midstream of two rivers (freshwater; n=10; salinity

Two newborn infants (one male and one female) were discovered dead and frozen in a home freezer. Although thawing is expected to lead to changes that may hamper postmortem investigations, the victims could not be examined in the frozen state and were thus immersed in saline at 37 degrees C to completely thaw them over about 90 min. Autolysis and putrefaction were not evident, postmortem changes were slight, and the internal organs were soft, allowing a thorough examination, including an autopsy and a histological investigation. Autopsy showed that both infants were full-term at the time of death. Hydrostatic tests of the lung and stomach indicated that the infants had been born alive, and based on histological analyses of pulmonary alveoli and bronchioles, they had started breathing. Malformations, pathological findings, and signs of suspected asphyxia were absent, and we assume that the infants were very likely to have been murdered. However, the cause of death is not yet fully understood. Although a police investigation revealed that the infants were smothered by occlusion of the mouth and nose and then frozen, there were no injuries on the skin around the nose and mouth. Nonetheless, we concluded that none of the postmortem findings were in conflict with this suspected mechanism of death. As these infants had been kept frozen since their death and thawed to body temperature as rapidly as possible in saline at 37 degrees C, their bodies were well preserved, which was helpful for the postmortem investigation.

We measured bacterioplankton in blood from cadavers retrieved from the sea ( n =12), near estuaries ( n =4), rivers (fresh water, n =8) and from bathtubs ( n =4) as well as from non-drowned victims ( n =10) discovered near aquatic environments. Blood from 11 victims drowned in seawater developed bioluminescent and/or blue colonies (oxidase test positive) on selective media containing 2–4% NaCl. Homology analyses of the 16S rRNA gene showed that all of them were marine bacteria (genera: Photobacterium , Vibrio , Shewanella , Psychrobacter ). Blood from all victims drowned in rivers generated blue colonies on plates containing ⩽3%, but not 4% NaCl. Homology analyses showed that the blue colonies were generated from bacteria that inhabit fresh water ( Aeromonas ). None of the blood samples from victims that drowned in bathtubs generated bioluminescent and blue colonies. However, all cadavers contained bacteria that produced unstained colonies ( Staphylococcus , Bacillus , Enterobacter , Escherichia , etc.). Among non-drowned victims, blood from two gave rise to blue colonies on plates containing ⩽3% NaCl ( Pseudomonas ). Of the cadavers found near estuaries, bioluminescent and blue colonies developed from two of them on media containing 2–4% NaCl ( Photobacterium , Vibrio , Listonella ), but not from two others on plates containing 4% NaCl (at ⩽3%; blue colonies, Aeromonas ; unstained colonies, Citrobacter , Vagococcus , Proteus , Enterobacter ). These results suggested that the presence of numerous bacterioplankton in immersed cadavers could support a conclusion of death by drowning.

We detected numerous bioluminescent bacteria in blood samples from two cadavers that had been immersed in estuarine environments. Autopsy, diatomaceous and toxicological findings indicated death by drowning, which agreed with environmental aspects and the findings of police investigations. Bioluminescent bacteria appeared in blood samples cultured on selective agar containing 2%, 3% and 4% NaCl after about 18h. Blood from the left side of the heart, the right side of the heart and the femoral vein generated 7.0 x 10(2), 2.0 x 10(4) and 8.0 x 10(2) cfu/ml of blood (case 1), and 1.8 x 10(4), 1.1 x 10(3) and 2.5 x 10(1) cfu/ml (case 2) of bioluminescent colonies, respectively, in agar containing 4% NaCl. Homologous analysis based on the 16S rRNA gene also identified the bioluminescent colonies as Vibrio fischeri and V. harveyi, which normally inhabit seawater. This simple assay might serve as an additional indicator to support a conclusion of death by drowning together with the diatom test.

We studied the effects of chronic alcohol intake on the disposition of alcohol and its metabolites in the rat. We used male Wistar rats for all of the experiments in this study. Using a pair-feeding process, rats were fed a liquid diet containing alcohol or without alcohol for 6 weeks. Ethanol solutions (0.5, 1.0, 1.5, and 2.0 g/kg body weight [BW]) were administered as a bolus, intravenously. We then measured blood ethanol and acetate concentrations. Simultaneous multiline fitting was performed using mean blood alcohol concentration (BAC)-time curves fitted to the one-compartment open model with parallel first-order and Michaelis-Menten elimination kinetics. At low doses (0.5, 1.0, and 1.5 g/kgBW), no differences were observed between the alcohol group and the control group with respect to ethanol elimination rate, area under the curve of ethanol (AUC(EtOH)), and mean residence time of ethanol (MRT(EtOH)). At higher doses (2.0 g/kgBW), ethanol elimination rate in the alcohol group was significantly higher than in the control group (P

We developed a method for genotyping Y chromosome-linked homologous DYS385 loci individually, combining locus-specific polymerase chain reaction (PCR) amplification and previously reported procedures. Duplicated DYS385a (5'-end) and DYS385b (3'-end) loci were located about 41 kb apart and inverted to each other in the Y chromosome, which data was obtained from the human genome sequence in National Center for Biotechnology Information (NCBI), and sequence differences were found at 424 bp down- and upstream of each locus. The locus-specific amplifications were performed using primers designed for this intergenic region, and fragments about 900 bp in length were produced. Polymorphic tetranucleotide arrays in the PCR products were typed according to procedures reported previously. Twenty male subjects were genotyped using this method. Alleles (GAAA)13-(GAAA)21 were observed at the DYS385a locus, and those at DYS385b included alleles (GAAA)9-(GAAA)15. DYS385a alleles, excluding those of identical arrays, were always larger than DYS385b alleles in the same subjects. These data suggest that the DYS385a and DYS385b loci can be amplified completely for discrimination, and the genotypes of the alleles provide information useful for forensic case work and population genetics.

Conventional acid digestion of tissues for analyzing diatoms obtained from suspected drowning cases is time-consuming, laborious and potentially dangerous. We propose a new protocol for solubilizing lung tissue using only Qiagen Proteinase K, Qiagen Buffer ATL, and 5N hydrochloric acid that can accelerate and simplify diatom extraction from suspected drowning cases. The lower lobe of the right lung (1g, inner region) and the upper lobe of the left lung (1g, peripheral region) of ten immersed victims were digested in 15-mL conical centrifuge tubes containing 9mL of Buffer ATL and 1mL of 20mg/mL Proteinase K solution at 56°C for 15-60min. The digest was washed with ultrapure water and then heated at 70°C for 15min in 5N hydrochloric acid. The acid residue was washed with ultrapure water followed by ethanol. The identification of 60-23,000 valves in 20 lung samples from the ten victims suggested that they had aspirated water before death. The proposed digestive protocol required only a low-speed centrifuge and a block incubator (or water bath) and diatoms could usually be extracted from lung samples within about 3h. Informative results of lung diatom tests were very helpful to confirm drowning as a cause of death. Therefore, the proposed protocol can be useful as a simple minimal test to support or challenge a diagnosis of death by drowning when characteristic autopsy findings of drowning are obvious and the probability of drowning is very high, when characteristic autopsy findings of drowning are absent and the probability of drowning is very low, and when conventional diatom testing is not performed due to logistic, personnel or budgetary limitations.

A 34-year-old man was discovered by his coworkers in a tank filled with 35% (w/w) hydrochloric acid. Despite undergoing intensive treatment, he died one and a half days later. An autopsy revealed generalized high tensity, overall grayish brown skin color, heavy gastric submucosal hemorrhage and heavy pulmonary edema. We concluded that death was caused by burn shock due to wide, generalized chemical burn. Microscopic investigation of the burn in the area with grayish brown skin considered coagulation necrosis of full-thickness of the skin (third-degree or deep burn), revealed that the burn was judged to cover the partial thickness of the skin (second-degree or dermal burn). These findings suggest that chemical burn by hydrochloric acid results in a change of skin color due to chemical reaction so that the appearance of the chemical burn is more severe than the degree assigned by histological examination.

The effects of baclofen and pancuronium bromide on evoked electromyogram (EMG), cortical electroencephalogram (EEG) and auditory brainstem responses (ABR) were studied in pentobarbital anesthetized normal rabbits. Evoked EMG was measured in the gastrocnemius muscle by electrical stimulation of the sciatic nerve. Intravenous injection of baclofen decreased EEG and arterial blood pressure and light reflex, however, it had no significant influence on EMG or ABR at doses of 10 and 20 mg/kg/h. Pancuronium bromide immediately inhibited respiration, decreased EEG and EMG, however, it had no significant influence on arterial blood pressure, ABR, or light reflex, at doses of 0.4 and 1.0 mg/kg/h in anesthetized rabbits. ABR waves were observed until just before cardiac arrest with both of the muscle relaxants. It is suggested that ABR are not influenced by central or peripheral muscle relaxants, or by pentobarbital.

Summary: Gc globulin, also called vitamin D-binding protein, is a human 56 kD plasma protein. Antigenic epitopes of the protein recognized by 12 different monoclonal antibodies (MoAbs), including the previously prepared MoAb El2 of which binding to Gc is inhibited by actin, were analyzed in relation to the functional domains of the protein. To map the epitopes recognized by the respective antibody, the reactivities of the MoAbs were tested by immunoblotting against the recombinant Gc fragments expressed in E. coli cells, and then a competitive ELISA was performed. The results showed that the antibodies were classified into at least six groups. Furthermore, in addition to El2, actin inhibited the reactivity of MoAb 21, of which the epitope was located within residues Asp32O to Glu 379, in an ELISA in the presence of the ligand. This result must indicate that this antibody-binding site is near the site that interacts with actin. In contrast, no inhibitory effect by 25-hydroxyvitamin D to any antibodies was observed. Furthermore, all antibodies, including El2 and 21, reacted with membrane-bound Gc of lymphocytes by an immunofluorescence study, suggesting that Gc is unlikely to bind to the plasma membrane in its interaction with actin.

A polyclonal bispecific (bifunctional) antibody was prepared to develop a hemagglutination immunoassay for beta-microseminoprotein (beta-MSP), a predominant seminal protein. Three types of F(ab')2 fragments of rabbit IgG, affinity-purified anti-human red blood cell (RBC) F(ab')2 nonaffinity-purified anti-beta-MSP F(ab')2 and nonspecific (nonimmunized) F(ab')2, were mixed to obtain a F(ab')2 mixture containing 10% anti-RBC molecules and 10% anti-beta-MSP molecules. Fab' was obtained from the F(ab')2 mixture, and then reacted with maleimide-activated bovine serum albumin (BSA) at a molar ratio of 10:1. As estimated by the decrease in the maleimide content, approximately 7 Fab' molecules were introduced per one BSA molecule. The bispecific (anti-beta-MSP and anti-RBC) Fab'-BSA conjugate thus prepared was incubated successively with a human RBC suspension and with samples. In the presence of beta-MSP, RBCs become agglutinated, providing a test simple for forensic semen identification.

Methemoglobin formation caused by a liniment solution containing sodium nitrite (30 g/L and 140 g/L) was studied in rats with normal or abraded skin, by measuring the methemoglobin concentration before and after application of liniment solutions with differing nitrite concentration.Each liniment solution (120 microL) was applied. Methemoglobin was measured for 180 minutes using a hemoximeter. Simultaneously, arterial blood pressure and cutaneous blood flow was measured by laser Doppler flowmetry and a pressure transducer.After the application of each liniment solution to normal skin, the methemoglobin concentration was not significantly modified depending on the time after application. Application of liniment solution to abraded skin (140 g/L) resulted in a marked increase in methemoglobin concentration. A remarkable decrease in arterial blood pressure and subcutaneous blood flow were observed after application of liniment solution to abraded skin (140 g/L).Each of these findings are characteristic of nitrite and they imply the percutaneous absorption of nitrite. Regardless of the nitrite concentration, the methemoglobin concentration was consistently higher in abraded skin than in normal skin.

Among many laboratory tests for supporting a conclusion of death by drowning, the diatom test is often regarded as the gold standard. However, opinions are divided on its usefulness, and thus this test is rarely or never applied in some countries or institutions. Indeed, even when characteristic macroscopic findings of drowning are weak, drowning can often be reasonably concluded as a cause of death by taking circumstantial evidence into consideration and carefully excluding other causes. However, incorporation of the diatom test will further increase the certainty of such a conclusion. When highly decomposed corpses are found in and near water environments, autopsy findings are of limited value, and the cause of death often remains undetermined. However, death that could have been due to drowning in some decomposed corpses can be assumed if the results of diatom testing are interpreted as positive. Pollanen illustrated the contribution of the diatom test to conclude drowning as a means of homicide in a presentation of six cases. The largely burned remains of a teenage girl were found in a parking lot where accelerant was detected. The results of an autopsy did not indicate the anatomical cause of death. However, 5 mL of watery fluid was aspirated from the right maxillary sinus. Over ten types of diatoms were detected in the fluid, and four of them that were also detected in bone marrow established drowning as the cause of death.

We developed a novel tool for concluding drowning as a cause of death. We designed nine primer pairs to detect representative freshwater or marine bacterioplankton (aquatic bacteria) and then used real-time PCR with TaqMan probes to rapidly and specifically detect them. We previously cultured the genus Aeromonas, which is a representative freshwater bacterial species, in blood samples from 94% of victims who drowned in freshwater and the genera Vibrio and/or Photobacterium that are representative marine bacteria in 88% of victims who drowned in seawater. Based on these results, we simultaneously detected eight species of bacterioplankton (Aeromonas hydrophila, A. salmonicida; Vibrio fischeri, V. harveyi, V. parahaemolyticus; Photobacterium damselae, P. leiognathi, P. phosphoreum) using three sets of triplex real-time PCR assays and TaqMan probes labelled with fluorophores (FAM, NED, Cy5). We assayed 266 specimens (109 blood, 157 tissues) from 43 victims, including 32 who had drowned in rivers, ditches, wells, sea or around estuaries. All lung samples of these 32 victims were TaqMan PCR-positive including the lung periphery into which water does not readily enter postmortem. On the other hand, findings in blood and/or closed organs (kidney or liver) were PCR-positive in 84% of the drowned victims (except for those who drowned in baths) although the conventional test detected diatoms in closed organs in only 44% of the victims. Thus, the results of the PCR assay reinforced those of diatom tests when only a few diatoms were detectable in organs due to the low density of diatoms in the water where they were found. Multiplex TaqMan PCR assays for bacterioplankton were rapid, less laborious and high-throughput as well as sensitive and specific. Therefore, these assays would be useful for routine forensic screening tests to estimate the amount and type of aspirated water.

We previously applied our method of detecting marine or freshwater bacterioplankton (bacteria) in the blood of immersed victims as a marker of drowning. However, we did not confirm the absence of post-mortem bacterial invasion during immersion. Here we examined the nature of bacterioplankton in blood samples from 21 immersed and 4 non-immersed cadavers. We found only freshwater bacterioplankton in the blood of two victims that were retrieved from the sea or an estuary inhabited by marine bacterioplankton even though one victim was highly putrefied. The results of diatom testing suggested that these two victims had drowned in fresh or brackish water with low salinity and then flowed out to the estuary or the sea. Two others were submerged in water, but representative bacterioplankton were undetectable in their blood although one victim was highly putrefied. Autopsy findings and the results of diatom tests did not indicate that the cause of death was drowning. As in previous studies, we identified freshwater bacterioplankton in the blood of seven other victims that had drowned in freshwater, marine bacterioplankton in the blood of four victims that had drowned in seawater and none in four victims found on land that had died by means other than drowning. Bacterioplankton in the blood of drowned victims appears to reflect the type of water aspirated and blood does not become easily contaminated with bacteria post-mortem even in decomposed bodies.

Glycyrrhetinic acid (GA), the main metabolic product of glycyrrhizin (GLY), could be detected in formalin-fixed tissue from a man who died 6 hours after therapeutic administration of a GLY-containing agent. GA was extracted from homogenized formalin-fixed liver tissue and 3 ng GA/g could be detected by HPLC. The extraction from formalin-fixed liver tissue gave the same retention time peak as the GLY control. GA could also be detected by mass spectrometry in the blood sample. This confirms that the man had received a GLY-containing agent for therapeutic use prior to his death and that GA can be determined from formalin-fixed tissue.

A decomposed female body with an open abdomen and pleural cavity washed up on a beach after a powerful typhoon. Autopsy findings could not determine the cause of death because of leaching and putrefaction. Numbers and types of diatoms in organs overall, suggested the aspiration of fresh or brackish water with low salinity. However, this could not be confirmed because of contamination via the open cavities. We simultaneously investigated the presence of bacterioplankton in liver, kidney and lung homogenates using a modification of our reported bacteriological method. The freshwater bacterioplankton Plesiomonas shigelloides was identified in each of these organs, but marine bacterioplankton were undetectable despite the circumstances under which the body was discovered. The presence of freshwater bacterioplankton reinforced the results of the diatom test, and we concluded that this victim had died of drowning in fresh or brackish water with low salinity.

To the Editor: The chambers of the great veins and the right side of the heart are frequently engorged not only in asphyxial deaths, but also in unquestioned natural deaths’)*). For the traditional reasons, in many Japanese forensic textbooks, however, the engorgement is described in a section of asphyxia with insufficient description of the association of engorgement with other types of deaths. Because of this, our students tend to assume that engorgement is a specific sign of asphyxia, and they often “comment upon” the engorgement whilst observing our postmortems (Fig. 1). To such students, we have been explaining that sudden cardiac arrest may be of itself sufficient to cause significant engorgement. In this letter, we

The g.ORF15 + 652-653delAG mutation in the RPGR gene is the most frequent mutation in X-linked retinitis pigmentosa (XLRP). The objective of this study was to investigate the possibility of mosaicism in an XLRP family. Eight subjects in the RP family were recruited. Blood samples were collected for DNA extraction. Haplotype analysis and mutational screening on the RPGR gene were performed. Additionally, samples of hair follicles and buccal cells from the mother of the proband were acquired for DNA extraction and molecular analysis. Phenotype was characterized with routine ophthalmic examination, Goldmann perimetry, electroretinography, and color fundus photography. A g.ORF15 + 652-653delAG mutation was identified in second- and third-generation patients/carriers. A first-generation female, who was considered to be an obligate carrier, demonstrated a normal phenotype as well as a normal genotype in lymphocytic DNA, indicating the gonadal mosaicism; however, a heterozygous AG-deletion at nucleotide 652 and 653 was identified in the genomic DNA of hair follicles, hair shaft, and buccal cells, indicating that the mutation is somatic. In conclusion, we reported on a family in which an asymptomatic woman with somatic-gonadal mosaicism for a RPGR gene mutation transmitted the mutation to an asymptomatic daughter and to a son with XLRP. Gonadal mosaicism may be responsible for a proportion of multiplex or simplex RP families, in which more than 50% of all cases of RP are found. (c) 2007 Wiley-Liss, Inc.

Accidental or suicidal poisonings due to benzodiazepines have been previously reported. A case of fatal, homicidal poisoning with benzodiazepines, antipyretic analgesics (anti-inflammatory drugs), and beer is described here. In this homicidal poisoning, the drugs and beer were given to the decedent by his wife. Autopsy findings showed no clinically significant macroscopic findings except for slight postmortem change. Capillary gas chromatography coupled to mass spectrometry was employed to quantitate the drugs in biological fluids and stomach contents. Six drugs (brotizolam, triazolam, ibuprofen, dihydrocodeine, phenylpropanolamine, and chlorpheniramine) were identified and quantitated in blood, urine, and stomach contents. Although each drug was present in a very small quantity, the cause of death was determined to be the combination of these drugs and alcohol poisoning. Language: en

CO-oximeters are specialized spectrophotometers that automatically determine hemoglobin (Hb) derivatives by measuring absorbance at selected wavelengths (1). We believe that a good understanding of the relevant theory may allow users to avoid many pitfalls during operation of these instruments. The mathematical basis of their operation has not, however, been fully explained by the manufacturers apart from Instrumentation Laboratory (Lexington, MA) at the introduction of their first CO-Oximeter™ (2). Here, we discuss what mathematical methods for data processing might be used in commercial CO-oximeters, particularly in those models that use an “overdetermined” system. CO-oximeters depend on the observation that Hb solutions obey the Lambert–Beer Law; thus, the absorbance measured at a given wavelength is the sum of the absorbance of each Hb derivative at the same wavelength (2). When we measure n wavelengths to determine the m Hb derivatives χ i , we get n equations: \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[\ A\_{j}{=}\ {{\sum}\_{i{=}1}^{m}}{\epsilon}\_{ji}C\_{i}l\mathrm{\ for\ 1}{\leq}j{\leq}n,\] \end{document} where A j is the absorbance at wavelength λ j , C i is the concentration of derivative χ i , and l is the pathlength. e ji is the molar absorptivity at wavelength λ j for derivative χ i . When n = m , we can solve Eq. 1 to get C i . This is termed an “exactly determined” system (3) and has been implemented in the IL 482 CO-Oximeter (Instrumentation Laboratory), the IL 282 (its predecessor), and the Radiometer OSM3 Hemoximeter™ (Radiometer, Copenhagen, Denmark). The IL 482 uses four wavelengths for four Hb derivatives, whereas the OSM3 uses six wavelengths for six unknowns: five Hb derivatives plus one for noise (attributed to “turbidity”). The report by Steinke and Shepherd (4) illustrates how the full exposition of the algorithms used in specific instruments is useful not only to users but also to manufacturers. After measuring absorptive spectra of Hb derivatives … [↵][1]bAuthor for correspondence. [1]: #xref-corresp-2-1

To investigate the effectiveness of marine bacteria as a new marker of drowning in seawater, we determined the optimal conditions of media required to selectively detect marine bacteria and applied the technique to drowned cadavers. We incubated model blood samples (n=20 per group) mixed with seawater, river, tap or muddy water on agar plates (Todd Hewitt, TH; Marine 2216, M2216) and determined the NaCl concentration required to selectively detect marine bacteria. We also used TCBS agar plates without manipulation to isolate Vibrio spp. Among the culture media, TH agar was superior. Bioluminescent colonies were detected only in blood mixed with seawater. Blue colonies stained using the cytochrome oxidase test (COT), were detected in blood mixed with both sea and river water. However when the NaCl concentration was above 4%, COT stained colonies were detectable only in blood mixed with seawater. We subsequently used 2, 3 and 4% NaCl in TH and TCBS agar to examine blood from victims who had drowned in seawater (n=8) and in fresh water (n=7), as well as from victims who died near aquatic environments (non drowned; dry-land control, n=7). Bioluminescent colonies were detectable on 2-4% NaCl TH agar only from two victims that drowned in seawater. Bioluminescent colonies did not grow on TCBS agar. Blue colonies from all cadavers that had drowned in seawater (8/8) and in four of those that had drowned in fresh water (4/7) proliferated on TH agar containing 2% and/or 3% NaCl, but at 4% NaCl such colonies were detected only from cadavers that had drowned in seawater (8/8). Colonies from only one cadaver from seawater grew on TCBS agar. Furthermore, neither bioluminescent nor blue colonies were detected on TH agar containing 4% NaCl in samples from two cadavers found in an estuary (brackish water) who were thought to have been carried from areas of fresh water. Homologous analyses of the 16S rRNA gene revealed that the dominant colonies on TH agar containing 4% NaCl were marine bacteria (Photobacterium, Vibrio, Shewanella, Psychrobacter). Thus, proliferating bioluminescent and/or blue colonies detected in the blood of immersed cadavers using 4% NaCl TH agar, could help to establish drowning in seawater.

A fatal case of methamphetamine poisoning whilst in police custody is described. Drug identification and quantification in postmortem blood, urine, brain and gastric contents was achieved using gas chromatography-mass spectrometry. The blood concentration of methamphetamine was 4.92 micromol/100 g, which is in the lethal range. It is necessary for police officers to have a good knowledge of the toxic effects of abused drugs.

Background : Plasma levels of remnant-like particles (RLP) is one of the predictive markers for coronary artery disease (CAD), and the inhibition by RLP of endothelium-dependent vasorelaxation has been reported. We attempted to clarify whether or not RLP, which inhibits endothelium-dependent vasorelaxation, affects nitric oxide (NO) production and NO synthase (eNOS) levels in cultured endothelial cells. Methods : RLP were obtained from postmortem blood of subjects who died of CAD. Modification by RLP of acetylcholine-induced relaxation of rabbit aorta, and changes in NO production and (eNOS) in cultured bovine endothelial cells were examined. Results : RLP at 750 and 1500 μg triglyceride/ml inhibited vasorelaxation, and at 5–160 μg triglyceride/ml, concentration-dependently inhibited NO production. However, (eNOS) did not decrease after incubation with RLP. Conclusion : Postmortem RLP from subjects who died of CAD do not change the amount of (eNOS), but rather, inhibits its activity and attenuates endothelium-dependent vasorelaxation.

The present study analyses the human Y-chromosome minisatellite locus, MSY1 (DYF155S1), in 205 Japanese males of 191 pedigrees using the minisatellite variant repeat (MVR) mapping system. The internal haploid structures of the detected alleles considerably varied and consisted of three major repeat units: types 2, 3 and 4. A comparison of the haploid profiles of the MVR codes identified 185 distinct alleles, of which only five were shared. We did not detect a type 1 repeat unit, and variations were frequent at the 5' end of the minisatellite locus. Within an analysis of 24 paternally linked DNA samples donated by ten families, no mutational events were identified even over two generation gaps. Furthermore, we applied this mapping system to a paternity test in which the alleged father was missing.

In our recent report, it remained unclear whether or not triglyceride-rich lipoprotein remnants (RLP) were associated with the risk of sudden coronary death in younger cases without coronary atherosclerosis that were detected in about 10% of all sudden coronary death cases in Japan. These cases were categorized as 'origin unknown, but suspected to be due to coronary spasm', the so called 'pokkuri disease' in Japan. The present study population consisted of 108 sudden death cases without coronary atherosclerosis [(pokkuri disease n=57) and non-cardiac sudden death (control n=51)] aged 20-69 years from Kanagawa prefecture in Japan. All individuals had died suddenly and unexpectedly, most had no significant history of medical conditions including cardiac symptoms and had not taken medications prior to death according to their medical records. All the autopsies were performed within 12 h after death. Plasma total cholesterol (TC), triglycerides (TG), phospholipids, RLP-C and RLP-TG, VLDL-C, LDL-C, HDL-C, apolipoproteins A-I, A-II, B, C-II, C-III, E, Lp (a) and homocysteine were measured in postmortem plasma samples. The TG-rich lipoprotein remnants measured as RLP-C and RLP-TG were significantly higher in pokkuri disease compared with controls both in fasting and postprandial states (P

Hitherto triglycerides (TG) and TG-rich lipoproteins were been of limited value as surrogates for antemortem levels. We measured TG levels in postmortem plasma from sudden coronary death cases (SCD, n=91) by using two TG assays, Dry Chem TG (free glycerol was added) and the Determiner L-TG (without added free glycerol) that measured net TG. TG levels were markedly higher by the Dry Chem TG (y) vs. Determiner L-TG (x), y=1.03x+229 mg/dl. HPLC showed large amounts of free glycerol in postmortem plasma and in TG-rich lipoprotein remnants (RLP). These results were verified in a rabbit model of SCD. Further, RLP from SCD were found to be biophysically similar to those from living patients with coronary artery disease (CAD). In conclusion, postmortem plasma sampled up to 12 h after death is appropriate for measuring lipid and lipoproteins, TG and RLP-TG as surrogates for antemortem levels when a TG assay without added free glycerol is used.

A 30-year-old male truck driver, known to be asthmatic, was found dead at the roadside lying near his car. A nebulizer bottle of Berotec (fenoterol hydrobromide) was found near his hand. The anatomic cause of death was suspected to be asthma. Toxicological screening of urine using Triage demonstrated the presence of methamphetamine. The blood concentration of methamphetamine was 0.4 microg/ml, and fenoterol was not detected. Hair analysis clearly indicated chronic methamphetamine abuse and medium dependency during the 2 months before death. We conclude that death might have been induced by the interaction of fenoterol and methamphetamine.

A case of fatal methemoglobinemia (MetHb-emia) which was presumably due to ingestion of nitrate is presented. An unidentified man was taken to a local emergency hospital suffering from deep cyanosis and 7 h later he was found dead in the hospital bed. The post-mortem examination of the blood revealed a methemoglobin (MetHb) concentration of 78% and the concentrations of nitrate and nitrite were 1.50 and 0.76 microg/mL, respectively. Capillary gas chromatography coupled to mass spectrometry (GC-MS) and capillary gas chromatography with a nitrogen-phosphorus detector (NPD) were used to detect nitrates and nitrites in the blood.

BACKGROUND: The "Back to Sleep" campaign in 1991 resulted in a dramatic decrease in the incidence of sudden infant death syndrome (SIDS). The proportion of presumed SIDS deaths being actually suspicious deaths from airway obstruction is likely to have become relatively greater. There is usually little pathological evidence to suggest smothering, but intra-alveolar haemorrhage appears to be more prominent in cases where interference with the airway is suspected. AIM: To attempt to quantify intra-alveolar haemorrhage to see whether it could be used as a marker to distinguish between smothering/overlaying and SIDS. METHODS: Intra-alveolar haemorrhage was quantified using digital image analysis on haematoxylin/eosin stained sections taken from the lungs of 62 consecutive infants who had died suddenly and unexpectedly. Cases were initially classified according to the original cause of death. After quantitation, the case histories were critically reviewed. Three pathologists independently made microscopic assessments of the degree of intra-alveolar haemorrhage in the first 24 cases to see whether these accurately reflected the quantitative results. RESULTS: 73% of those infants with a history suggesting possible involuntary overlaying and 45% of those with a history suspicious of smothering had significant intra-alveolar haemorrhage (> 5% of total lung surface area assessed). From the history, the cause of death in 11 cases initially classified as SIDS would better have been given as "Unascertained." Simple microscopic assessments underestimated the true extent of the haemorrhage in 33% (8/24). CONCLUSIONS: If a moderate degree (at least 5%) of pulmonary parenchymal haemorrhage is observed, this may be an indicator of airway obstruction for a significant period, either from overlaying or possibly smothering. The diagnosis of SIDS may be being used inappropriately in such cases.

Among the risk factors for coronary atherosclerosis, elevated LDL-C level is best known. The action of lipoprotein lipase on triglyceride-rich lipoproteins produces remnant lipoprotein particles enriched in cholesterol and apolipoprotein E (apo E). Apo E serves as the ligand for uptake of remnant lipoproteins via the LDL-receptor or the remnant receptor. In this study, postmortem plasma total cholesterol, triglycerides (TG), VLDL-C, HDL-C, lipoprotein (a) [Lp(a)] and remnant-like lipoprotein particles (RLP)-cholesterol, RLP-TG, apolipoproteins B, C III and E were measured, together with LDL-C to assess their potential contribution to the severity of coronary and aortic atherosclerosis of the 197 cases of sudden death (132 cardiac death and 65 non-cardiac death). In all cases, the severity of coronary atherosclerosis was determined at postmortem pathological examination. RLP-cholesterol (RLP-C) and LDL-C concentrations were significantly higher in cases with advanced coronary atherosclerosis compared with those without coronary atherosclerosis; respective median values were 13.5 vs 8.4 mg/dl (P < 0.001) and 140 vs 115 mg/dl (P < 0.05). RLP-C levels were more strongly correlated with the severity score of coronary atherosclerosis than LDL-C.

Gc globulin binds actin at an equimolar ratio with a high affinity. It has been revealed that complexes of Gc with actin are present in the plasma of human cadavers. In the present study, an immunoassay which we developed using a specific anti-Gc monoclonal antibody was applied to evaluate the complexed Gc in cadaver plasma. The percentage of Gc in the complexed form was found to be 38.3 +/- 19.8% (n = 45), and a significantly higher percentage was observed in the plasma of cadavers from burn death. However, the plot of the complex percentage against the time since death showed a relatively low correlation, with a coefficient (r) of 0.51. This was supposed to be due to different environmental conditions after death. Since the complex formation represents the release of cytoplasmic actin into plasma from collapsed cells, the phenomenon should be noted in forensic postmortem chemistry.

Alpha 2 -HS glycoprotein (AHSG) is a human plasma glycoprotein and fetuin is the homologue in the calf. In this report, we present the structure and organization of the AHSG gene. Introns and the 5′ and 3′-flanking regions were obtained by polymerase chain reaction (PCR) and the inverted PCR, respectively, from genomic DNA using AHSG cDNA-specific oligonucleotide primers. The sequence of the PCR products shows that the coding region spans approximately 8.2 kb and is composed of seven exons interrupted by six introns. The exon–intron splice junctions agree with the consensus sequence, and the positions interrupted by introns are precisely identical to those of the rat insulin receptor tyrosine kinase inhibitor (fetuin) gene. The 5′-promoter region contains several characteristic sequences such as an A+T-rich sequence of TAAATAA, C/EBP-binding site, and hepatocyte nuclear factor-5 (HNF-5) and serum response factor (SRF) sites.