Bone Molecular Modifications Induced by Diagenesis Followed-Up for 12 Months.

After death, diagenesis takes place. Numerous processes occur concomitantly, which makes it difficult to identify the diagenetic processes. The diagenetic processes refer to all processes (chemical or physical) that modify the skeletal remains. These processes are highly variable depending on the environmental factors (weather, temperature, age, sex, etc.), especially in the early stages. Numerous studies have evaluated bone diagenetic processes over long timescales (~millions of years), but fewer have been done over short timescales (between days and thousands of years). The objective of the study is to assess the early stages of diagenetic processes by Raman microspectroscopy over 12 months. The mineral and organic matrix modifications are monitored through physicochemical parameters. Ribs from six humans were buried in soil. The modifications of bone composition were followed by Raman spectroscopy each month. The decrease in the mineral/organic ratio and carbonate type-B content and the increase in crystallinity reveal that minerals undergo dissolution-recrystallization. The decrease in collagen cross-linking indicates that collagen hydrolysis induces the fragmentation of collagen fibres over 12 months.

Critical aspects of Raman spectroscopy as a tool for postmortem interval estimation.

The estimation of the postmortem interval (PMI) from skeletal remains represents a challenging task in forensic science. PMI is often influenced by extrinsic factors (humidity, dryness, scavengers, etc.) and intrinsic factors (age, sex, pathology, way of life, medical treatments, etc.). Raman spectroscopy combined with multivariate data analysis represents a promising tool for forensic anthropologists. Despite all the advantages of the technique, Raman spectra of skeletal remains are influenced by these extrinsic and intrinsic factors, which impairs precision and reproducibility. Both parameters have to reach a high level of confidence when such spectroscopy is used as a way to predict PMI. As a consequence, advanced multivariate data analysis is necessary to quantify the effect of all factors to improve the estimation of the PMI. The objective of this work is to evaluate the effect of intrinsic and extrinsic factors on the Raman spectra of skeletal remains. We designed a protocol close to a real-world scenario. We used ANOVA-simultaneous component analysis (ASCA) to unmix and quantify the effect of 1 intrinsic (source body) and 1 extrinsic (burial time) factors on the Raman spectra. In our model, the burial time was found to generate the highest variability after the source body. ASCA showed that the variability due to the burial time has 2 mixed contributions. Seasonal variations are the first contribution. The second contribution is attributed to diagenesis. A decrease in the mineral bands and an increase in the organic bands are observed. The source body was also found to contribute to the variability in Raman spectra. ASCA showed that the source body induces variability related to the composition of bones. This quantification cannot be assessed by basic chemometrics methods such as PCA. The results of this study highlighted the need to use an advanced chemometric data analysis tool (like ASCA) combined with Raman spectroscopy to estimate the postmortem interval.

An overview of forensic operations performed following the terrorist attacks on November 13, 2015, in Paris.

On the evening of November 13, 2015, the city of Paris and its surroundings was hit by a series of attacks committed by terrorist groups, using firearms and explosives. The final toll was 140 people deceased (130 victims and 10 terrorists or their relatives) and more than 413 injured, making these attacks the worst mass killings ever recorded in Paris in peacetime. This article presents the forensic operations carried out at the Medicolegal Institute of Paris (MLIP) following these attacks. A total of 68 autopsies of bodies or body fragments and 83 external examinations were performed within 7 days, and the overall forensic operations (including formal identification of the latest victims) were completed 10 days after the attacks. Over this period, 156 body presentations (some bodies were presented several times) were provided to families or relatives. Regarding the 130 civilian casualties, 129 died from firearm wounds and one died from blast injuries after an explosion. Of the 10 terrorists or their relatives who were killed, eight died from suicide bombing, one was shot by police and one died from crush injuries due to partial collapse of a building following the police raid against a terrorist's hideout after the attacks. All mass shootings were perpetrated with AK-47 or Zastava M70 assault rifles using 7.62 mm × 39 mm cartridges. In the case of ballistic injuries, death was most often obviously caused by craniocerebral injuries, extensive organ lacerations and/or massive haemorrhage. Among the terrorists killed by bombing, the lesion patterns were body transection, multiple amputations, extreme organ lacerations and the presence of foreign bodies owing to the shrapnel load (steel nuts, glass fragments) or the explosive charge fastening system of the devices. This discussion highlights the particular difficulties of interpretation encountered within the framework of ballistic injuries, a conclusion that should lead to a modest and realistic approach in these exceptional situations where forensic operations involve a very large number of victims in a constrained time.

Terrorist attacks: cutaneous patterns of gunshot and secondary blast injuries.

Terrorist attacks have been on the rise. During the recent terrorist attacks in France, terrorists perpetrated their acts using weapons of war, as well as explosive charges. These two modes of action, when combined, can create skin lesions with similar macroscopic appearances, which can sometimes go unnoticed because of body fragmentation. A total of 68 autopsies, 83 external examinations, 140 standard radiographic examinations, and 49 computed tomography (CT) scans were performed over 7 days during the 2015 terrorist attacks in France. Bodies were injured by firearms and shrapnel-like projectiles. We analysed the clinical findings for the secondary blast cutaneous lesions from the explosive devices and compared these lesions with ballistic-related lesions to highlight that patterns can be macroscopically similar on external examination. Secondary blast injuries are characterised by penetrating trauma associated with materials added to explosive systems that are propelled by explosive air movement. These injuries are caused most often by small, shrapnel-like metallic objects, such as nails and bolts. Propulsion causes ballistic-type injuries that must be recognised and distinguished from those caused by firearm projectiles. Differentiating between these lesions is very difficult when using conventional criteria (size, shape, number and distribution on the body) with only external examination of corpses. This is why the particularities of these lesions must be further illustrated and then confirmed by complete autopsies and radiological and anatomopathological examinations.Key pointsWhen occurring simultaneously in terrorist attacks, injuries caused by secondary blasts appear as cutaneous wound patterns that can be macroscopically very similar to those caused by firearm projectiles.The criteria usually found in the literature for distinguishing these two types of projectiles may be difficult to use.It is important in these difficult situations to benefit from systematic postmortem imaging.Systematic autopsy and then anatomopathological analyses of the orifices also help determine the cause of the wounds.

Determination of the age of bruises using a bilirubinometer.

The dating of bruises can be of crucial interest in clinical forensic medicine, but the macroscopic or photographic evaluation of a bruise has not been regarded as reliable. Several methods have thus been regularly evaluated for this purpose, with reflectance spectrometry yielding promising results. The objective of this study is to evaluate the value of a bilirubinometer as a tool for dating bruises on living victims. A clinical follow-up was carried out on patients presenting a bruise with a known onset. Using a bilirubinometer, we obtained daily measurements of the bruise (bili-bruise) and of healthy skin (bili-skin). Potential confounding factors were collected: age, sex, body mass index, trauma mechanism and Fitzpatrick skin phototype. We followed 20 patients for a total of 88 measurements of bruises. Bili-skin values showed significant differences according to skin phototype. Differences between the bili-bruise and bili-skin values (Δ-bili) followed an increase phase, peaking between 3 and 5 days, and then there was a decrease phase. No significant Δ-bili value differences were observed based on the suspected confounding factors. Our results are in favour of a peak Δ-bili value generally at day 4 or 5 post-trauma. Notably, decreasing values were not observed before day 3. Decreasing Δ-bili values would then indicate a bruise resulting from an injury formed at least 3 days before the first measurement. Complementary work confirming such data would enable improvement of the performance of bruise dating in forensic medicine.

Terrorist explosive belt attacks: specific patterns of bone traumas.

In cases of terrorist bomb attacks, the issue of victim identification is the principal aim. For investigators and justice, terrorists must also be identified in a timely manner. The fragmentation of bodies caused by explosive devices however makes this operation difficult. The cases presented correspond to six autopsies of perpetrators carrying explosives. Their explosive belts produced different body sections on areas correlated with the location on the body of carrying explosive charges. Indeed, cross-sectional areas are the signature of the level of explosive devices on the body, and some fracture features through the damage of certain areas of constitutional weaknesses are specific elements to consider. These elements can allow a quick distinction between victims and terrorists, in order to assist in the medico-legal process of identification.

Characterization of bone diagenesis by histology in forensic contexts: a human taphonomic study.

The diagenesis of a bone in the postmortem period causes an identifiable deterioration in histology. This degradation is characterized by a collagenous alteration, which can be observed very early. In order to develop a method for determining a postmortem interval for medico-legal use, two ribs collected from six human bodies were studied prospectively over 2 years. Each bone was studied after staining with Sirius red to demonstrate the degradation of collagen as a function of time. This study demonstrated a time-based bone alteration characterized by the architectural degradation of the lamellar bone, without any microbial influence in this postmortem period. The staining was carried out by using Sirius red and correlated this alteration with a collagenic degradation by chemical hydrolysis owing to the affinity of this dye to the amino acids lysine, hydroxylysine, and arginine. Our work asserts that human bone samples that were studied in a controlled environment and analyzed for 24 months underwent a diagenetic trajectory whose main element was collagen hydrolysis.

Domestic Predation of an Elder: A Fatal Dog Attack Case.

We present the case of a 91-year-old woman lived alone at her home with two domestic dogs, that is,, a Labrador Retriever and a Staffordshire Bull Terrier and found dead. The investigation of the scene revealed that the Bull Terrier's jawbone and chest were covered with blood. The autopsy revealed multiple, histologically confirmed, life-threatening skin and bone lacerations without scavenging marks. The punctures and tearing of each of the wounds on the skin were compatible with bites. A left humeral fracture and multiple fractures of the right facial bones were observed. The death was attributed to external hemorrhages due to several dog bites. A veterinary physical and behavioral examination indicated that the Bull Terrier was involved in the attack. A domestic predation hypothesis was deemed here most likely due to the presence of food supplies at the scene, the dog's previous history of attack, and the breed of the dog.

Correction: New Insights on the Composition and the Structure of the Acellular Extrinsic Fiber Cementum by Raman Analysis.

[This corrects the article DOI: 10.1371/journal.pone.0167316.].

The precision of micro-tomography in bone taphonomic experiments and the importance of registration.

Micro-computed tomography (µCT) provides micrometric 3D images and has been used in forensic studies for anthropology pubis measurement or insect description for post mortem interval estimation. Studies have suggested using registration, a superimposing images method between a reference and a target. This technique avoids positioning bias and increase the precision of µCT. However, no clear study has reported the precision with µCT analysis before or after registration in a forensic field. One fresh post mortem sample of a human cranial vault was collected. Two successive µCT acquisitions (resolution 10µm) of it were performed without repositioning. The data from the second acquisition were copied and registered by two trained operators (operators 1 and 2). Operator 1 performed a second registration process after 1 week (operator 1 bis). The images were analysed. The bone volume (BV), bone surface (BS), number of trabeculae (TbN), trabecular thickness (TbTh) and mean trabecular distance (TbSp) were compared before and after registration. The mean (±SD), the coefficient of variation (%CV), and the precision error of the standard deviation absolute value and of the coefficient of variation between operators 1 and 2 (inter-subject variability) and between operator 1 and 1 bis (intra-subject variability) were calculated. We also collected the second phalanx of the 2nd, 3rd and 4th fingers on the hands of a second individual. Two successive scans (resolution 27µm) were performed without repositioning. A comparison (mean±SD of BV, BS, TbN, TbTh, TbSp) was made between the first and second scans with and without registration, and an ANOVA repeated measures procedure was performed. For the vault, we show that after 30 registrations for each operator (1, 2 and 1 bis), the mean and %CV were very close for each variable and between operators. For BV and BS, the difference in the mean value was approximately 0.01 (mm3 and mm2, respectively). The precision error was higher in the inter-subject registrations for each variable. The precision error magnitude for all variables was very low (<0.01) in absolute value and of %CV. For the fingers, the difference between the first and second scans may be approximately 50% without registration. We found that the second scan without registration is significantly different for BV (p=0.006), BS (p=0.007), TbN (p=0.019) and TbSp (p=0.002). Knowing the precision of the device (with and without registration) is important to ensure that the accuracy of the µCT results.

Technical note: early post-mortem changes of human bone in taphonomy with µCT.

Post-mortem interval (PMI) estimation is an important issue in forensic medicine, particularly for criminal purposes and legal limitation periods. The goal of the present study is to examine the evolution of the trabecular cranial vault bone after 4 weeks of conservation in a controlled environment with micro-tomography (µCT) analyses.Four bone samples were extracted from a fresh human cranial vault (a donation to science according to the French law) and conserved in an air-controlled environment. The samples were weighed and µCT scanned at a 10-µm resolution every week after death for a month. The µCT features were identical for every sample. Each set of data from the µCTs was reconstructed, registered, and analyzed in terms of the total volume, bone volume, bone surface, number of trabeculae, trabeculae thickness, and mean distance of the trabeculae. The samples were conserved in a glass box in 20 °C air with 60% humidity in a laboratory hood between each µCT acquisition. Descriptive statistics were determined. Each sample was observed and compared to itself over time.After 1 month of conservation, the mean bone volume (-1.9%), bone surface (-5.1%), and trabecular number (-12.35%) decreased, whereas the mean trabecular separation (+5.55%) and trabecular thickness (+12.7%) increased. Many variations (i.e., increases and decreases) were observed between the extraction of the sample and the end of the 4 weeks of conservation. The present observations may be explained by bone diagenesis. Previous observations have indicated that protein and lipid losses occur with bone weight and volume losses. These diagenesis effects may explain the trabecular modifications observed in the present work. We observed many bone variations with the µCT scans between the beginning and the end of the conservation that had no explanations. Additional studies, particularly studies involving statistics, need to be performed to confirm our observations and explain these results more clearly.

New Insights on the Composition and the Structure of the Acellular Extrinsic Fiber Cementum by Raman Analysis.

Acellular extrinsic fiber cementum is a mineralized tissue that covers the cervical half of the tooth root surface. It contains mainly extrinsic or Sharpey's fibers that run perpendicular to the root surface to anchor the tooth via the periodontal ligament. Acellular cementum is continuously and slowly produced throughout life and exhibits an alternating bright and dark pattern under light microscopy. However, although a better understanding of the structural background of acellular cementum is relevant to many fields, such as cementochronology, periodontology and tissue engineering, acellular cementum remains rarely studied and poorly understood. In this work, we studied the acellular cementum at the incremental line scale of five human mandibular canines using polarized Raman spectroscopy. We provided Raman imaging analysis and polarized acquisitions as a function of the angular orientation of the sample. The results showed that mineral crystals were always parallel to collagen fibrils, and at a larger scale, we proposed an organizational model in which we found radial collagen fibers, "orthogonal" to the cementum surface, and "non-orthogonal" fibers, which consist of branching and bending radial fibers. Concerning the alternating pattern, we observed that the dark lines corresponded to smaller, more mineralized and probably more organized bands, which is consistent with the zoological assumption that incremental lines are produced during a winter rest period of acellular cementum growth.

Cyanide Suicide After Deep Web Shopping: A Case Report.

Cyanide is a product that is known for its use in industrial or laboratory processes, as well as for intentional intoxication. The toxicity of cyanide is well described in humans with rapid inhibition of cellular aerobic metabolism after ingestion or inhalation, leading to severe clinical effects that are frequently lethal. We report the case of a young white man found dead in a hotel room after self-poisoning with cyanide ordered in the deep Web. This case shows a probable complex suicide kit use including cyanide, as a lethal tool, and dextromethorphan, as a sedative and anxiolytic substance. This case is an original example of the emerging deep Web shopping in illegal drug procurement.

Organ Procurement in Forensic Deaths: Specific Features of Nonheart-Beating Donors.

The need for organs has increased in the recent years, and this shortage will only worsen. In addition to the organs procured from brain-dead donors, a procedure for non-heart-beating donors was therefore developed. When deaths involve legal proceedings, the medical examiner makes the decision of whether or not to remove organs. To assist medical examiner in their decision-making, a number of forensic scientific societies aimed to develop recommendations, and legal adjustments were adopted. Nevertheless, these do present certain limits in special cases of procurement: Maastricht class I and II non-heart-beating donors. The peculiarity of this procedure stems from the urgency of the process. Compliance with temporal criteria would require that the deceased subject be removed from the crime scene even before the arrival of the authorities. This is extremely problematic legally and technically because forensic teams must collect evidence at crime scenes. Developments will have to be made to further minimize the restrictions on donations in forensic deaths.

Effects of the environment on bone mass: A human taphonomic study.

An experiment was designed using human ribs placed in different environments to document how moisture and temperature affect the bone mass according to the postmortem interval. The bones were defleshed, weighed and partially buried, with some ribs being left unburied as controls. The ribs were weighed daily, and the mass loss was monitored over a period of 90days. The results showed that significant differences in bone mass loss exist between environments, where the bone mass loss was significantly faster in an environment with low moisture content. This mass loss is thought to be primarily associated with the desiccation of the body and then for a greater part, with the atmospheric moisture content. However, the loss of bone mass can also be explained by early alterations in the organo-mineral matrix which were highlighted by Raman spectroscopy method.

Bone Samples Extracted from Embalmed Subjects Are Not Appropriate for the Assessment of Bone Quality at the Molecular Level Using Raman Spectroscopy.

Bone samples extracted from embalmed cadavers are commonly used as controls in the study of bone. The effects of embalmment on the molecular composition of bone are unknown. The objective of this study was to determine the effect of embalmment on the molecular composition and structure of bone, as evaluated by Raman spectroscopy. Bone samples of femoral heads from five embalmed donors and five fresh-frozen donors were compared using Raman microspectroscopy with DuoScan technology. Physicochemical parameters simultaneously describing the organic and mineral phases of bone were compared using the Mann-Whitney U test. Partial least squares discriminant analysis (PLS-DA) was used to determine specific Raman spectral features of each group. Study of the mineral phase showed a 15% reduction of the mineral-to-matrix ratio (p < 0.001), an 8% decrease of type B carbonate substitution (p < 0.001), and a 2% increase in crystallinity (p < 0.001) in the embalmed donors group compared to those of the fresh donors group. Regarding the organic phase of bone, the hydroxyproline-to-proline ratio was increased by 18% in the embalmed group (p < 0.001), with no variation in both the relative proteoglycan content (GAG/CH3) (p = 0.08) and collagen maturity (p = 0.57). PLS-DA showed that the embalmed group was characterized mainly by peaks assigned to hydroxyproline, lipids, and collagen. Embalmment induces significant modifications of the molecular composition of bone. Bone samples from embalmed subjects should be avoided as controls for Raman spectroscopy studies. Preservation procedures performed prior to bone sampling should be reported in studies using human cadaver samples.

The mechanism of the keyhole lesion reassessed: An experimental approach.

The initial description of the keyhole defect was detailed as a peculiar gunshot entrance wound in the cranial vault due to firearm discharge in a tangential path. This injury may be described in two parts: a rounded section with inner table beveling and a triangular section with outer table beveling. We report a case of a gunshot skull wound "keyhole" shaped, appeared to have been made perpendicularly to the bone. Performing an experimental study on cranial bones with shots made perpendicularly to the skull approved this hypothesis, and bone injuries were then architecturally characterised using high-resolution micro computed tomography. The singular link between the tangential gunshot path and the keyhole pattern has been refuted several times, and some authors have hypothesised that there is an important role for concentric fractures that occur far away from the initial impact point of the bullet. Micro computed tomography analysis of the bone showed these keyhole defect features with a particular high description. Then, the whole pattern has a spider-web effect, and these concentric fractures could explain the keyhole pattern even in a perpendicular gunshot path.

Organ procurement in forensic deaths: French developments.

Organ procurement and transplantation have grown steadily, and the need for organs will only rise in the future. Increasing the number of potential donors is therefore paramount. However, transplant coordination teams face refusals that can be linked to the contexts of the deaths, especially when they involve legal issues. In France, deaths involving legal proceedings are not uncommon (7-10%). In these cases, the prosecutor is immediately contacted, and makes the decision of whether to remove the involved organs. Refusals of this type represent 4% (approximately 30 cases per year) of obstacles to organ removals, and are governed by specific legislation. Thus, the prosecutor must arrange contact with a forensic pathologist and with the organ transplant teams to assemble all of the necessary elements for him to take the decision. To assist prosecutors in their decision making and to ensure them scientific rigour, the French Society of Forensic Medicine sought to develop a national recommendation to harmonise practices; it emerged in early 2013. The guideline makes practical recommendations, including among others: nominating local referents; writing regional protocols between judicial authorities, forensic pathologists and transplant teams; establishing terms for the forensic pathologist's intervention on the donor's body before and after a procurement. This recommendation by the French Society of Forensic Medicine aimed to combine two interests: addressing the shortage of organs, and fulfilling the requisites of a criminal investigation by standardising practices and encouraging communication.

Homemade Firearm Suicide With Dumbbell Pipe Triggering by an Air-Compressed Gun: Case Report and Review of Literature.

Firearm suicides are frequent and well described in the forensic literature, particularly in Europe and the United States. However, the use of homemade and improvised firearms is less well described. The present case reports a suicide with an original improvised gun created using an air-compressed pellet gun and a dumbbell pipe. The aims of this study were to describe the scene, the external examination of the corpse, the body scan, and the autopsy; to understand the mechanism of death; and to compare the results with a review of the forensic literature to highlight the epidemiology of homemade firearm use, the tools used for homemade and improvised firearms in suicides versus homicides, and the manners in which homemade firearms are used (homicide or suicide, particularly in complex suicide cases).

Experimental study of Lucilia sericata (Diptera Calliphoridae) larval development on rat cadavers: Effects of climate and chemical contamination.

Household products such as bleach, gasoline or hydrochloric acid have been used to mask the presence of a cadaver or to prevent the colonization of insects. These types of chemicals affect insect development and alter the forensic entomology analysis. This study was designed to test the effects of six household products (bleach, mosquito repellent, perfume, caustic soda, insecticide and unleaded gasoline) on blowfly (Lucilia sericata, Diptera: Calliphoridae) larval development. Furthermore, the effects of climate (rain or dry conditions) on larval development were analyzed. For each replication, 100 first instars were placed on a rat cadaver on which one household product was spilled. We observed a decrease in the survival rates of the larvae but no significant effect on their development times or the adult size. The same trends were observed under rainy conditions. However, the rain altered the effects of some tested household products, especially gasoline. These results demonstrate for the first time the successful development of necrophagous larvae on chemically contaminated cadavers, and provide evidence for the range of possible effects to expect.