A method for bone marrow extraction of diatoms for forensic science applications.

Diatoms show potential as trace evidence indicators, particularly as evidence of drowning. Often, the diatom test to diagnose drowning is done on soft tissue or bone marrow from a recently deceased individual. This method presented here combines elements from previous forensic literature and methods of diatom isolation in phycology to extract diatoms from bone marrow of skeletal remains for forensic use. This diatom extraction method is time-efficient, minimizes contamination risk, and produces samples of intact diatoms. This method is designed to complete sample preparation within 24 h, sampling the bone for diatoms internally and externally. This method was developed using porcine long bones submerged in water with live diatoms for up to 3 months. Three marrow samples were extracted from each bone so the method was developed using 102 marrow samples. Additionally, 132 surficial bone and environmental samples were collected and prepared during method development. To briefly summarize the method, the bone joints were cut off with an angle grinder in a biosafety hood to expose the marrow, which was removed from the hip, knee, and shaft as separate samples. The marrow was digested with nitric acid at 400°C in glass beakers before being centrifuged with DI water, plated onto microscope slides, and observed with a compound microscope. Observation found good preservation of unbroken diatom cell walls throughout the process. This method can be used to prepare diatoms as forensic trace evidence.

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.

Potential forensic application of receptor for advanced glycation end products (RAGE) and aquaporin 5 (AQP5) as novel biomarkers for diagnosis of drowning.

Drowning is the most common cause of unnatural death worldwide. There is no single biomarker to diagnose drowning, so the diagnosis of drowning is one of the most difficult tasks in forensic medicine. Especially, distinguishing a victim of drowning from a body disposed of in water following death remains a problem. The objective of this study was to identify specific biomarkers of drowning compared with other causes of death such as hypoxia and postmortem submersion. The present study investigated the intrapulmonary expression of receptor for advanced glycation end products (RAGE), aquaporin-5 (AQP5), surfactant protein-A (SP-A), interleukin 6 (IL-6) and interleukin 1ß (IL-1ß) as markers of drowning. In animal experiments, all rats (n = 45) were classified into four groups (drowning, postmortem-submersion, hypoxia and control group). The lungs of experimental animals were analyzed as mRNA expression, immunoblot expression and immunohistochemical staining. qRT-PCR demonstrated increased mRNA expression of RAGE and AQP5 in drowning group compared with that in control, hypoxia and postmortem-submersion group, but not other molecules. Western blotting also showed high expression of RAGE and AQP5 in drowning group, immunostaining of RAGE and AQP5 was highly detected in a linear pattern in type I alveolar epithelial cells, compared with control and postmortem-submersion group. These observations indicate a difference of expression in pulmonary molecular pathology compared with other causes, suggesting RAGE and AQP5 may be useful for differentiation between drowning and postmortem-submersion.

The diatoms test in veterinary medicine: A pilot study on cetaceans and sea turtles.

Fishing activities are considered one of the most relevant threats for cetaceans and sea turtles conservation since these animals are sometimes found dead entangled in fishing gears. Currently, postmortem diagnosis is based mainly on the presence of nets and lines on the body and the related marks and injuries evident at gross examination. A more detailed and objective evidence is needed to clarify doubts cases and the diatoms technique, used in forensic human medicine, could support drowning diagnosis also in this field. Diatoms' investigation was implemented to be applied in marine vertebrate on 8 striped (Stenella coeruleoalba) and 1 bottlenose (Tursiops truncatus) dolphins and 5 sea turtles (Caretta caretta) stranded along the Italian coastlines with a likely cause of death hypothized on necropsies carried out by veterinary pathologists. Diatoms were microscopically searched in the bone marrow collected from long bones implementing protocols used in human medicine and their presence was observed in 4 cetaceans and 2 sea turtles. Despite a clear relation between diatoms' presence and amount and the likely cause of death was not proved due to the poor number of samples, the higher burden of diatoms was found in 3 animals deemed to be death for the interaction with human activity. Despite more studied are necessary to identify the possible relation between the cause of death and diatoms' findings, the present study implemented this technique to be adapted to marine animals, confirming its possible application also in veterinary forensic medicine.

Complex Suicide by Self-stabbing and Drowning: A Case Report and a Review of Literature.

Sharp force injuries and drowning are methods exceptionally combined in complex suicides. We report a challenging case of complex suicide by self-stabbing and drowning that illustrates the difficulty in discriminating between homicide and suicide in such circumstances of death. The corpse of a young man was found submerged in a river, stabbed nine times with two wounds that had penetrated the thorax and had caused lung injuries and a hemopneumothorax. The postmortem and histological examinations were consistent with a death caused by drowning, but the manner of death still remained undetermined. Police investigation finally concluded to a suicide, although no suicide note had been left and the victim had no underlying diagnosed mental disorder. The parameters that may help distinguish suicide from homicide at the autopsy should be interpreted in light of a thorough forensic investigation to determine the exact manner of death in such a case.

A valid method to determine the site of drowning.

The diatom test is considered a useful aid in determining the site of death from drowning. Nevertheless, there is disagreement within the scientific community concerning its reliability, and its findings have been challenged and sometimes overturned in courts of law. Using a model based on animal experimentation, we have developed a diatom test to discriminate between the locations of drowning sites from different aquatic systems. We carried out a complementary combination of quantitative and qualitative analyses together with a statistical analysis based on the Kullback-Leibler distance of the samples. A restrictive selection of exclusive diatom species from each reservoir was also made. This approach allowed us to validate the usefulness of the diatom test in determining the location of the site of drowning.

Understanding scuba diving fatalities: carbon dioxide concentrations in intra-cardiac gas.

INTRODUCTION: Important developments in the diagnosis of scuba diving fatalities have been made thanks to forensic imaging tool improvements. Multi-detector computed tomography (MDCT) permits reliable interpretation of the overall gaseous distribution in the cadaver. However, due to post-mortem delay, the radiological interpretation is often doubtful because the distinction between gas related to the dive and post-mortem decomposition artifactual gases becomes less obvious. METHODS: We present six cases of fatal scuba diving showing gas in the heart and other vasculature. Carbon dioxide (CO2) in cardiac gas measured by gas chromatography coupled to thermal conductivity detection were employed to distinguish decomposition from embolism based on the detection of decomposition gases (hydrogen, hydrogen sulfide and methane) and to confirm arterial gas embolism (AGE) or post-mortem offgasing diagnoses. A Radiological Alteration Index (RAI) was calculated from the scan. RESULTS: Based on the dive history, the intra-cadaveric gas was diagnosed as deriving from decomposition (one case, minimal RAI of 61), post-mortem decompression artifacts (two cases, intermediate RAI between 60 and 85) and barotrauma/AGE (three cases, maximal RAI between 85 and 100), illustrating a large distribution inside the bodies. CONCLUSION: MDCT scans should be interpreted simultaneously with compositional analysis of intra-cadaveric gases. Intra-cadaveric gas sampling and analysis may become useful tools for understanding and diagnosing scuba diving fatalities. In cases with short post-mortem delays, the CO2 concentration of the cardiac gas provides relevant information about the circumstances and cause of death when this parameter is interpreted in combination with the diving profile.

Post-mortem computed tomography coaxial cutting needle biopsy to facilitate the detection of bacterioplankton using PCR probes as a diagnostic indicator for drowning.

We report for the first time the use of coaxial cutting needle biopsy, guided by post-mortem computed tomography (PMCT), to sample internal body tissues for bacterioplankton PCR analysis to investigate drowning. This technical report describes the biopsy technique, the comparison of the needle biopsy and the invasive autopsy sampling results, as well as the PMCT and autopsy findings. By using this new biopsy sampling approach for bacterioplankton PCR, we have developed on previous papers describing the minimally invasive PMCT approach for the diagnosis of drowning. When such a system is used, the operator must take all precautions to avoid contamination of the core biopsy samples due to the sensitivity of PCR-based analytic systems.

Evaluation of two methods for the use of diatoms in drowning cases.

In this study, we compare digestive methods used in a forensic context to extract diatoms (37 % hydrochloric acid) to a method recently described in Italian protocols for analysis of benthic diatoms for ecological assessment of surface water (hydrogen peroxide digestion). The two digestive methods were performed using 5 g of brain, lung, liver, kidney, and bone marrow taken from the bodies of 10 drowning victims recovered from three different aquatic environments (ocean, lakes, and rivers). Postmortem examination was performed on all bodies, but aquatic samples were only analyzed in two cases. Tissue digestion was equal by both methods. Hydrogen peroxide (H2O2) digestion resulted in better diatom preservation, enabling identification of nine genera in all samples examined versus three obtained using hydrochloride digestion. The ideal digestive method to provide evidence for corroboration of a diagnosis of drowning still needs to be established. However, the benthic diatoms protocol can be useful because it is less chemically hazardous to the laboratory operator and supports better diatom preservation for reliable taxonomic analysis.

[Drowning versus cardiac ischemia: Cardiac arrest of an 11-year-old boy at a swimming lake]. / Ertrinken vs. kardiale Ischämie: Elfjähriger Junge mit Herz-Kreislauf-Stillstand am Badesee.

This report describes a case of sudden cardiac arrest and subsequent attempted cardiopulmonary resuscitation of an 11-year-old child on the shores of a swimming lake. Reports of eyewitnesses excluded the obviously suspected diagnosis of a drowning accident. The result of the autopsy was sudden cardiac death due to a congenital coronary anomaly (abnormal left coronary artery, ALCA). Favored by vigorous physical activity, this anomaly can lead to malignant arrhythmias because the ectopic coronary artery with its intramural course through the aortic wall is compressed during every systole. This pathology was not known to the boy or his family; in fact he liked sports but had suffered of a syncope once which was not followed up. Without a strong suspicion it is difficult to diagnose a coronary artery anomaly and it is often missed even in college athletes. Tragically, sudden cardiac arrest may be the first symptom of an undiagnosed abnormal coronary artery. Following syncope or chest pain during exercise with a normal electrocardiogram (ECG) cardiac imaging, such as computed tomography (CT) or angiography should be initiated in order to enable surgical repair of an abnormal coronary artery.

Application of the microwave digestion-vacuum filtration-automated scanning electron microscopy method for diatom detection in the diagnosis of drowning.

The cause of death for the decomposed corpses recovered from water is still a difficult issue in current forensic practice. In this article, we present two cases of bodies recovered from water with no positive findings to indicate the cause of their death. We apply both conventional acid digestion method as well as the microwave digestion-vacuum filtration-automated scanning electron microscopy method (MD-VF-Auto SEM) for diatom detection in different organs of both bodies. Our results indicate that MD-VF-Auto SEM method provide more accurate information and match further police investigation. This novel method would be a useful technique in assessing cause of death for body found in water.

Svechnikov's sign as an indicator of drowning in immersed bodies changed by decomposition: an autopsy study.

PURPOSE: Bodies recovered from water often present as a difficult problem in forensic pathology. The aim of this study was to examine the presence and amount of free liquid in the sphenoid sinus in cases of freshwater drowning, and to compare this to the amount found in putrefied bodies recovered from freshwater, as well as in putrefied bodies found in an indoor environment. METHODS: Free liquid from the sphenoid sinuses was aspirated using a syringe and a needle, after piercing the hypophyseal fossa. Non-putrefied drowning cases were also examined for hemolytic staining of the intima of the aortic root. RESULTS: In 29 non-putrefied cases of freshwater drowning there was 1.36 ± 1.48 ml in the sphenoid sinuses, with 21 of them having hemolytic staining of aortic intima. In putrefied bodies recovered from freshwater (22 cases) there was 1.26 ± 1.40 ml within the sphenoid sinuses, and in putrefied bodies found in an indoor environment (52 cases), there was significantly less-0.57 ± 0.92 ml. CONCLUSIONS: Free liquid in the sphenoid sinuses (Svechnikov's sign) may be considered a vital reaction in drowning non-putrefied cases. Hemolytic staining of the aortic intima could be a significant sign of freshwater drowning. In putrefied bodies recovered from water, an amount of 0.55 ml of free liquid in the sphenoid sinuses may imply that the victim was alive upon their contact with the water, but the presence of free liquid in the sphenoid sinuses does not necessarily indicate that drowning had been the cause of death.

Selective culturing and genus-specific PCR detection for identification of Aeromonas in tissue samples to assist the medico-legal diagnosis of death by drowning.

The detection of autochthonous aquatic bacteria in tissue samples from drowning cases is increasingly considered as an alternative approach to assist the medico-legal diagnosis of death by drowning. Bacteria belonging to the genus Aeromonas may be suitable candidates for this application as they are ubiquitous in natural aquatic environments but are generally not part of the human microbiota. The research aims of this study were (i) to develop a sensitive, specific and rapid screening and confirmation method for Aeromonas species in tissue samples and (ii) to evaluate aseptic sternal puncture as a post-mortem sample technique and bone marrow as an alternative matrix to provide evidence of death by drowning. The presence of Aeromonas in tissue samples was verified by cultivation using the selective media Ampicillin Dextrin Agar (ADA) and Ryan's Aeromonas Medium. The use of ADA medium was found most optimal for the sensitive, inexpensive and quick detection of aeromonads in human tissue samples. Positive culture plates were confirmed by harvesting all colonies for DNA extraction and subsequent PCR amplification using Aeromonas genus-specific primers. Aeromonads were detected in lung swab, blood and bone marrow of drowned bodies (n=3), but were negative in these three matrices for all negative controls (n=90) tested. Bone marrow proved to be a suitable alternative matrix and can be sampled post-mortem by an aseptic sternal puncture. In conclusion, this study confirms previous indications that aeromonads in cultures from blood of water bodies can be considered a potential marker for drowning. Given the fact that the number of immersed bodies (drowned and non-drowned) included in this study is statistically not significant, however, more tissue samples need to be investigated to confirm the validity of these methods to aid the diagnosis of death by wet drowning.

Trial investigation of post-mortem non-invasive transnasal endoscopy.

We performed a trial investigation of transnasal endoscopy for post-mortem examinations to assess its efficacy in superficial post-mortem examinations. Transnasal endoscopy proved capable of permitting detailed visual inspections of the respiratory and the upper gastrointestinal tract, equal to direct viewing, on an LCD digital display. In 18 (40.9%) of 44 cases, findings obtained by post-mortem transnasal endoscopy (PMTNE) provided valuable clues regarding cause of death. The cases examined included seven deaths by fire, four by hypothermia, four by asphyxia (three involving death by mechanical asphyxia, the other by choking), two by drowning and one case of lung cancer. In two cases, PMTNE also led to informative findings not directly related to the cause of death. Under Japan's current systems, we are required to diagnose the cause of death for over 80% of all the unnatural death cases based solely on superficial post-mortem examinations, in the absence of an autopsy. Introducing PMTNE to superficial post-mortem examinations will undoubtedly provide much more information on the cause of death than relying solely on superficial post-mortem examinations.

The potential significance of elevated vitreous sodium levels at autopsy.

Elevated levels of sodium that may be detected in the analysis of post-mortem fluid samples may arise from a wide variety of organic illnesses and environmental factors that have caused either water depletion or solute gain. When hypernatraemia is suspected at autopsy a careful history is required with particular emphasis on pre-existing medical conditions such as renal or gastrointestinal disease. In addition, detailed information is required on the circumstances of death, including any clinical symptoms and signs that the deceased may have manifested, or medical procedures such as dialysis, colonoscopy or intravenous fluid replacement. Reduced intake of fluid may be associated with cognitive or physical impairment or may result from environmental depletion (the latter may be a manifestation of inflicted injury). Both central and nephrogenic diabetes insipidus may result in the loss of excessive amounts of hypotonic fluid. This may also occur following diuretic use, or be due to gastroenteritis or burns. Hypernatraemia may be a marker of excessive salt/saline ingestion and/or administration and may occur accidentally or as a manifestation of child abuse. Given the range of possible etiologies, hypernatraemia may be a significant autopsy finding that requires explanation.

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 degrees 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.

Forensic implication of muscle level of nicotine in an adipocere body found in the sea.

We reported previously that blood levels of nicotine in suicidal smokers tend to be significantly higher than those in non-suicidal smokers, and blood level of nicotine seems to be a useful criterion for discriminating suicide from other types of death. In this paper, we report nicotine and cotinine levels in various tissues of an adipocere body found in the sea. The cause of death was drowning, and the postmortem time interval was approximately 5 months at autopsy. His driver's license was concealed in his sock, which seemed to suggest that he committed to suicide. In toxicological analysis by gas chromatography, nicotine and cotinine in the femoral muscle were detected at concentrations of 213 and 488 ng/g, respectively, and these substances were also detected in the brain, liver and kidney. For evaluating the tissue levels of nicotine and cotinine in the adipocere body, we analyzed these levels in blood and various tissues of 13 autopsy cases of smokers. Nicotine and cotinine levels in blood were the most similar to those in skeletal muscle. Although the postmortem time interval, the formation process of adipocere and the environmental condition in water may affect nicotine and cotinine levels in the femoral muscle, the high muscle level of nicotine in the present case seem to implicate suicidal death.

Detection of diatom in formalin-fixed organs by enzymatic digestion.

The detection of diatoms has been proposed to be useful in the diagnosis of drowning. Enzymatic digestion of unfixed lungs and other organs is widely used to detect diatoms. In the present study, we examined the application of enzymatic digestion for diatom detection in formalin-fixed organs obtained at autopsy. Furthermore, we assessed the effect of hydrogen peroxide on contamination of the lung specimen with foreign bodies inhaled in the course of drowning or as a result of smoking or air pollution. Unfixed lung was digested with proteinase K for 2d and centrifuged; the supernatant was then removed. On the other hand, formalin-fixed lung samples were boiled in Tris-HCl buffer containing sodium dodecyl sulfate (SDS) and then subjected to enzymatic digestion. A portion of formalin-fixed or unfixed samples digested with proteinase K was incubated with hydrogen peroxide at 80 degrees C and mounted on a slide, while the remaining portion was processed without incubation. Samples incubated with hydrogen peroxide showed clearer image of diatoms than those not incubated with hydrogen peroxide; this is because the inhaled foreign bodies were discolored in the former samples. Therefore, incubation of digested samples with hydrogen peroxide would be helpful for quantitative and qualitative analyses of diatoms.

Development and standardization of a microalgae test for determining deaths by drowning.

The diatom test for the diagnosis of drowning is widely used in countries of the Northern Hemisphere such as France and Japan. In Latin America, however, it has not been adopted as a routine procedure in forensic autopsies. In aquatic ecosystems, dinoflagellates and some chlorophytes are microalgae that, like diatoms, have cell walls and other resistant structures. As a result, they can be found in tissues from drowning victims, which is important because diatoms may be rare under particular environmental conditions. On this basis, we propose to extend the "diatom test" to include other microalgae for the determination of death by drowning. In this work, we developed a standardized procedure for detecting microalgae in tissues from drowning victims, with techniques described in the international literature and designed by us. The corpses were recovered from coastal areas in Antofagasta Region, Chile, during summer 2005. The most effective procedure for the treatment of water and tissue samples involved the combination of enzymatic digestion (proteinase K) and chemical digestion. The technique allowed the recovery of dinoflagellate evidence belonging to genera Prorocentrum, Ceratium, Dinophysis and Protoperidinium; silicoflagellates of the genus Dictyocha; an undetermined, filamentous chlorophyte; entire valves of centric diatoms and fragments of pennate and centric diatoms. This is the first protocol using microalgae other than diatoms for forensic cases in Latin America, and particularly in Chile.