Technical note: Excel spreadsheet calculation of the Henssge equation as an aid to estimating postmortem interval.

In forensic cases for which the time of death is unknown, several methods are used to estimate the postmortem interval. The quotient (Q) defined as the difference between the rectal and ambient temperature (Tr - Ta) divided by the initial difference (T0 - Ta) represents the progress of postmortem cooling: Q = (Tr - Ta)/(T0 - Ta), (1 ≥ Q ≥ 0). Henssge was able to show that with the body weight and its empirical corrective factor, Q can be reasonably predicted as a double exponential decay function of time (Qp(t)). On the other hand, actual Q is determined as Qd by measuring Tr and Ta under an assumption of T0 = 37.2 °C. Then, the t value at which Qp(t) is equal to Qd (Qd=Qp(t)) would be a good estimate of the postmortem interval (the Henssge equation). Since the equation cannot be solved analytically, it has been solved using a pair of nomograms devised by Henssge. With greater access to computers and spreadsheet software, computational methods based on the input of actual parameters of the case can be more easily utilized. In this technical note, we describe two types of Excel spreadsheets to solve the equation numerically. In one type, a fairly accurate solution was obtained by iteration using an add-in program Solver. In the other type (forward calculation), a series of Qp(t) was generated at a time interval of 0.05 h and the t value at which Qp(t) was nearest to Qd was selected as an approximate solution using a built-in function, XLOOKUP. Alternatively, a series of absolute values of the difference between Qd and Qp(t) (|Dq(t)| = |Qd - Qp(t)|) was generated with time interval 0.1 h and the t value that produces the minimum |Dq(t)| was selected. These Excel spreadsheets are available as Supplementary Files.

A Suggested Mechanism for Green Discoloration of the Postmortem Brain.

ABSTRACT: In the putrefied brain, the cortex and basal ganglia show dark-grayish to green discoloration due to sulfhemoglobin formed from hydrogen sulfide (H 2 S) produced by endogenous bacteria and hemoglobin. In this study, we propose and demonstrate another mechanism of green discoloration in the brain. The formalin-fixed brain of a cadaver donated for medical education with no putrefaction was used. Half of the brain was immersed in sodium hydrosulfide solution, to imitate the H 2 S produced by bacteria. This half showed greenish discoloration, mainly in the basal ganglia and cortex. The other half showed positive Perls' Prussian blue staining, mainly in the basal ganglia and cortex. The area of greenish discoloration due to H 2 S and the region positive for Perls' Prussian blue staining coincided. Tissue treatment with strong oxidizing agents is required to liberate heme iron. The positive Perls' Prussian blue staining in this study thus does not reflect heme iron. In conclusion, we considered that non-heme iron compounds physiologically present in the brain and H 2 S represent sources of putrefactive greenish discoloration in the brain.

Hemorrhagic shock due to ruptured idiopathic intramural hematoma of the sigmoid colon-An autopsy case report.

We describe herein an autopsy case involving fatal hemorrhagic shock due to idiopathic sigmoid colonic intramural hematoma rupturing into the abdominal cavity. Antemortem computed tomography revealed a mass lesion in the sigmoid colon and a large amount of hemoperitoneum. On forensic autopsy, intramural hematoma of the sigmoid colon with ruptured serosa was identified, while the mucosa remained intact. Microscopically, hematomas were apparent, mainly in the muscularis propria. We diagnosed the cause of death as hemorrhagic shock due to idiopathic intramural hematoma of the sigmoid colon rupturing into the abdominal cavity. Although the patient had been receiving peritoneal dialysis, no relationship was identified between dialysis catheters and sigmoid colon intramural hematoma rupture. On computed tomography, the mass lesion was initially considered a submucosal neoplastic lesion or endometriotic lesion. Intramural hematoma should be considered as a differential diagnosis.

Conventional diatom testing using strong acid: (II) Number and types of diatoms detected in closed organs and lungs of 80 autopsy cases using only new Kjeldahl flasks.

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.

An autopsy case report of tramline bruises with various shapes -histological and mechanistic considerations.

An autopsy case involving a child showing various shapes of tramline bruises caused by a single weapon is described. On autopsy, multiple tramline bruises in different orientations were found, mainly on the back. Shapes of the tramline bruises were variable, including linear, arcuate, nodular, hook-like, and hook-like with a knot at the end. Due to the presence of severe subcutaneous hemorrhages and the absence of other potentially fatal injuries or diseases, we determined the cause of death to be traumatic shock. Microscopically, of the many tramline bruises, characteristic hemorrhages were observed on the right abdominal and left lumbar regions. Those hemorrhages were observed in the dermis, corresponding to macroscopic tramline bruises. Hemorrhage was also observed in the subcutis, corresponding to the pallor of the outer surface. The mechanisms by which tramline bruises occurred in the dermis and subcutis in this case are discussed with reference to the literature. We also propose subcutaneous and intradermal hemorrhagic types of tramline bruising.

A Case of Fatal Exsanguination by a Japanese Short Sword.

ABSTRACT: We report a rare case of fatal exsanguination by a wakizashi , a Japanese short sword ( shinken ). A man in his 60s was found lying on his back on his bed. He was in cardiopulmonary arrest when the ambulance crew arrived and was pronounced dead on arrival at the hospital. A wakizashi with blood stains on the blade was found on the floor, and blood was present on the bed sheets on which the man was found. Autopsy revealed an incised wound extending diagonally from superolaterally to inferomedially on the anterior left thigh as the only injury. The wound appeared mostly straight, approximately 44 cm long and 9 cm deep, with smooth walls. The left femoral artery and vein had been cleanly severed, and the cause of death was determined to be blood loss from the severed blood vessels. The wakizashi (blade length, 45 cm) was large enough to have caused the wound, and the left thigh was presumed to have been incised by a downward slash from the sword.

Conventional diatom testing using strong acid: Notable false-positive results caused by an underestimated contamination source (blind spot).

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 SiO2, similar to glass. Adherent diatoms are then released from the glass by re-heating at the next diatom testing. These results also suggest that the number of diatoms remaining in a flask can increase steadily as a result of repeated reuse for analysis of lung or water samples. In contrast, in analyses using only new flasks, only one or two diatoms were found in 4 of 20 kidney, 2 of 12 liver, and 2 of 8 blood samples from 20 drowning victims. It is difficult to determine whether such diatoms are actually carried via the blood circulation, as contamination with a few diatoms can occur during autopsy procedures and diatom testing. In conclusion, only new (unused) Kjeldahl flasks should be used for diatom testing with strong acid digestion. Moreover, these data suggest that the number and frequency of diatoms present in closed organs of drowning victims may be much lower than previously thought.

"Black ring-shaped burn" in button battery ingestion is not a burn - Comparison with charring using spectral CT.

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.

Adult Dismembered Body With Myositis Ossificans: Evidence for Physical Abuse in an Autopsy Case Report.

ABSTRACT: 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.

Acceleration effect of the forensic luminol reaction induced by visible light irradiation of whole human blood aqueous solutions.

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.

A new enzymatic method for extracting diatoms from organs of suspected drowning cases using papain: Optimal digestion and first practical application.

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.

Simple detection of bacterioplankton using a loop-mediated isothermal amplification (LAMP) assay: First practical approach to 72 cases of suspected drowning.

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 80min 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.

Autopsy findings for a case of acute gastric volvulus in a child.

Acute gastric volvulus resulting in abdominal compartment syndrome was determined to be the cause of death in a 4-year-old girl who presented with abdominal distension. At about 1AM on the day of her death, she was brought to our emergency medical center. Physical examination and plain abdominal X-ray revealed pronounced gastric dilatation. A decompression procedure was performed, followed by observation. She went into cardiopulmonary arrest around 1PM on the same day and died. Postmortem investigation, including an autopsy and computed tomography (CT), was performed to determine the cause of death. The findings included that the stomach was severely distended. Evidence was seen of mucosal hemorrhage in the gastric mucosa on the greater curvature side, which was thinned in places but without perforation. No necrosis of the gastric mucosa was observed; reversible changes were evident on histopathological examination. The postmortem CT images suggested that the pyloric region was positioned cranioventrally to the cardiac region. None of the findings indicated sudden blockage, and the cause of death was determined to be acute gastric volvulus resulting in abdominal compartment syndrome. The abnormal placement of the organs was difficult to determine based on physical examination alone; postmortem CT and careful examination were helpful in conducting the autopsy in this case.

Simple protocol for extracting diatoms from lung tissues of suspected drowning cases within 3h: First practical application.

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 new molecular approach to help conclude drowning as a cause of death: simultaneous detection of eight bacterioplankton species using real-time PCR assays with TaqMan probes.

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.

Detection of diverse aquatic microbes in blood and organs of drowning victims: first metagenomic approach using high-throughput 454-pyrosequencing.

Current 454-pyrosequencing technology enables massive parallel sequencing. We used this technology to investigate the diversity of aquatic microbes in 14 specimens (blood and organs) of two drowning victims and in two water samples taken from the discovery sites. The 16S ribosomal RNA (rRNA) genes of microbes, which are often used to identify species (or genera), have nine highly variable regions (V1-V9), each of which is surrounded by conserved regions. Some parts within the conserved regions are common over domains of microbes, such as between bacteria and algae (16S rRNA genes on algal chloroplast genomes). We therefore simultaneously amplified the target regions (V7 and V8) of various microbes in the blood and organs of drowning victims using PCR with custom-designed primers that were based on the conserved regions. We then exhaustively analyzed the PCR products by pyrosequencing using the Genome Sequencer FLX Titanium system (Roche-454 Life Sciences). This approach identified a wide array of bacteria including cyanobacteria and algae including Bacillariophyceae (diatom), Cryptophyceae, Dictyochophyceae, Chrysophyceae and Trebouxiophyceae in the blood and organs of the victims and water at discovery sites. Our data further indicated that when conventional diatom testing of lungs yielded insufficient evidence of water aspiration, the detection of various exogenous microbes by 454-pyrosequencing is very useful to support a conclusion of death by drowning. To the best of our knowledge, this is the first attempt to use a new generation sequencer to investigate diverse aquatic microbes in the blood and closed organs of drowning victims.

Numbers, sizes, and types of diatoms around estuaries for a diatom test.

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.

Detection of marine and freshwater bacterioplankton in immersed victims: Post-mortem bacterial invasion does not readily occur.

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.