A field study to evaluate PMI estimation methods for advanced decomposition stages.

Estimating the postmortem interval (PMI) is one of the major tasks and a continuous challenge in forensic pathology. It is often an exclusion process of available methods, which ultimately can lead to an unsatisfactory outcome due to poor reliability. This problem is most acute in the late PMI, when decomposition proceeds and some methods (such as rigor, livor, and algor mortis) are no longer applicable. Several methods, such as forensic entomology, skeletal muscle protein degradation, and the study of body decomposition by application of a morphological scoring, are expected to provide further information; however, all have certain limitations and weaknesses. Availability of a tool-box of methods allows a case-specific selection of the most appropriate one(s), or eventually provides improvements in the overall accuracy and precision of the PMI estimation by merging and combining methods. To investigate practical (field) application, eventual interferences, and/or synergetic effects, as well as the robustness of these methods towards specific influencing factors, a field study was conducted, using eight pig cadavers of different body weights and physical coverage, left to decompose under natural conditions for 16 days. Morphological changes during decomposition were assessed using the total body score (TBS), muscle samples were collected to analyze protein degradation, and insect colonization was evaluated. The results reveal strengths and current limitations of all tested methods, as well as promising synergistic effects, and thus, provide a baseline for targeted future research.

Diatoms in drowning cases in forensic veterinary context: a preliminary study.

In this preliminary study, a digestive method used in forensic context to extract diatoms has been applied in organs taken from ten wildlife animals belonging three species of mammals (a deer, a roe and five otters) and one species of birds (two magpies). Only four carcasses were recovered from aquatic environments (bath trough for animals, bathtub irrigation, river) and only in three cases out of ten that the cause of death was ruled out as drowning. In seven cases, the death was due to other causes: gunshot injuries for one otter, blunt trauma for a magpie, and traumatic injuries followed by motor vehicle collision in other four otters and a roe. Post-mortem examination was performed in all carcasses. The diatom test protocol was performed according to the Italian guidelines for analysis of benthic diatoms for ecological status assessment of inland waters. Five grams of lung, liver, and kidney was taken from all the animal carcasses. In some cases, additional tissue samples were also available among which brain, heart, spleen, and bone marrow. In all four cases found in water, the drowning medium was also available. Hydrogen peroxide (H2O2) digestion was performed, and identification of 21 genera of diatoms was obtained. The method proved to be suitable for the identification of diatoms in the organs of the animals drowned supporting the final diagnosis of drowning. Only in otters, all died for causes other than drowning, diatoms did not prove to be suitable for the diagnosis of drowning since their presence in the internal organs was mainly related to their main diet based on fishmeal. The authors believe that this first trial is very promising, and the results suggest that diatom test can be easily applied in forensic veterinary context.

Pupillary effects in habitual cannabis consumers quantified with pupillography.

Driving under the influence of alcohol (DUIA) and drugs (DUID) is considered an elevated risk for traffic safety. When assessing a driver's fitness to drive, standardized and objective measurement methods are still required, in order to clarify the question whether an individual is under the influence of substances acting on the central nervous system (CNS). We exposed healthy test subjects (n=41) as well as persons who were under the influence of cannabis after repeated inhalation to multiple light stimuli using infrared technology and measured the pupillary light reflex (PLR). Toxicological tests of blood samples taken from every subject followed. The aims of this study were to assess the differences in pupillography response between cannabis consumers after a washout period and no cannabis consumers as well as the dose related effects on pupillography parameters of cannabis in cannabis consumers. All four pupillary parameters changed according to a weakened pupil function after acute administration of cannabis in all test subjects. Furthermore, it could be observed that habitual cannabis consumers showed an altered pupillary function just before the first dose was taken, suggesting that the long-term effects and addiction also have to be taken into account, when effects of the CNS are discussed. The results of the present study show that almost all pupil parameters could be reliable indicators for the detection of subjects under the acute effect of cannabis.

The applicability of forensic time since death estimation methods for buried bodies in advanced decomposition stages.

Estimation of the postmortem interval in advanced postmortem stages is a challenging task. Although there are several approaches available for addressing postmortem changes of a (human) body or its environment (ecologically and/or biochemically), most are restricted to specific timeframes and/or individual and environmental conditions. It is well known, for instance, that buried bodies decompose in a remarkably different manner than on the ground surface. However, data on how established methods for PMI estimation perform under these conditions are scarce. It is important to understand whether and how postmortem changes are affected under burial conditions, if corrective factors could be conceived, or if methods have to be excluded for respective cases. We present the first multi-methodological assessment of human postmortem decomposition carried out on buried body donors in Europe, at the Amsterdam Research Initiative for Sub-surface Taphonomy and Anthropology (ARISTA) in the Netherlands. We used a multidisciplinary approach to investigate postmortem changes of morphology, skeletal muscle protein decomposition, presence of insects and other necrophilous animals as well as microbial communities (i.e., microbiomes) from August to November 2018 associated with two complete body exhumations and eight partial exhumations. Our results clearly display the current possibilities and limitations of methods for PMI estimation in buried remains and provide a baseline for future research and application.

The mummified corpse in a domestic setting.

A mummified body of an 86-year-old white man with a history of coronary atherosclerosis was found in the reception-room of his apartment located in a condominium of the city-center of Bari (Southern Italy) approximately 7 years after disappearance. Unpaid electricity bills caused the power stations to turn off electricity while unpaid condominium bills forced the manager of the condominium to open the apartment where the body was found. The corpse was well preserved through the mummification process and no external injuries were observed. Signs of a very low insect activity were also present, reasonably consistent with a rapid skin dehydration. The body was sitting on the carpet in front of an easy-chair, fully clothed by a woollen vest with dark paints and shoes. The head was lying face down on the easy-chair. The corpse was very light in weight, fixed in the sitting position by the brittle dehydrated tissues. The carpet on which the body was sitting and the covering tissue of the easy-chair on which the head was lying absorbed most of the early putrefactive fluids coming from the corpse. The internal organs were grossly identifiable but essentially unremarkable. On histologic examination most of the tissues were found to be autolyzed. Toxicology studies revealed only decompositional products. The cause of death was undetermined but presumed natural. Based on the ante-mortem data available the corpse was soon identified by dental comparison to be that one of the apartment owner missing 7 years before. The delayed recovery of elderly people who lived alone, incapacitated or unable to get help, or even of lonely deaths of nobody seems to miss are often explained by the isolation os such people even in urban areas in addition to a deficient family support, missing social and neighborly relationships, worsening of the health and financial conditions.

Postmortem artifacts made by ants and the effect of ant activity on decompositional rates.

Ants belong to the family Formicidae of the order Hymenoptera and they are one of the world's dominant insect groups. Ants can be present at all stages of carrion decomposition as they are typically observed shortly after death or during the early postmortem period, but even later once the fly maggots had left the body. Their role in the faunal succession varies from predator on the eggs and larvae of other insects (mainly Diptera), thus reducing significantly the rate of decomposition, to scavenger on the flesh or exudates from the corpse itself. As they can deeply affect the occurrence of cadaver entomofauna, the presence or absence of ants should be taken into account in every case involving postmortem interval estimates based on entomological evidence. In fact, the effects of ants on carrion reduction seem to be mainly dependent on species, on their amount or abundance, and on geographic area. The feeding action of ants can cause many irregular, serpiginous, scalloped areas of superficial skin loss, and small punctate and scratch-type lesions may be often observed on the body, which are the result of postmortem ant bites. Usually ant injuries are orange-pink to yellow in color and diffusely scattered over the skin surface. These injuries consist of small and rather shallow gnawed holes that can be easily misinterpreted as antemortem abrasions or resulting from strong acids. No bleeding is associated with such skin lesions but sometimes considerable hemorrhage can take place, especially where removal of superficial layers of skin occurs in congested parts of the body. As ants attack the uncovered areas of the body, ant bites can frequently give rise to suspicion especially if located on the neck mimicking antemortem injuries. They are also occasionally misinterpreted as patterned abrasion due to the imprinted effect of a blunt or offending object. On closer inspection, artifacts made by ants can be immediately apparent especially when the ants are identified upon the body, but final diagnosis can be only confirmed at the autopsy by gross and microscopic analysis. The features of such lesions will be illustrated in detail by reviewing some interesting forensic cases.

Fatal adverse reaction to ketorolac tromethamine in asthmatic patient.

A case of an asthmatic woman who collapsed within a few minutes after intramuscular ketorolac tromethamine (KT) injection is reported. Autopsy findings revealed anatomic evidence of a recent asthma attack. KT was found to be present in the blood at a concentration within the therapeutic range and consistent with the administered dose. Based on the timing of the collapse in relation to the KT administration, death was attributed to an adverse reaction to KT, resulting in acute bronchospasm and cardiac arrest, with asthma as an underlying contributing factor. In this case, asthma alone was not responsible for the death of the patient but only a contributing factor. Physicians have to be aware that in asthmatic patients bronchospasm can be induced by drugs among which aspirin or nonsteroidal anti-inflammatory drugs such as KT are the most common; therefore, death may have an iatrogenic cause. The paper also describes the pathogenic mechanism of an adverse reaction to such drugs and analytical methods for the isolation and detection of KT in postmortem blood.

Sudden cardiac death and myocardial ischemia indicators: a comparative study of four immunohistochemical markers.

The postmortem diagnosis of acute myocardial infarction represents a current challenge for forensic pathologists, particularly when death occurs within minutes to a few hours after the ischemic insult. Among the adult population the single most important cause of sudden cardiac death (SCD) is the well-known atherosclerotic coronary artery disease, commonly asymptomatic or unrecognized. The recognition of early myocardial damage using routine hematoxylin and eosin (H&E) staining is possible only if death has occurred at least 6 hours after the onset of the ischemic injury. The usefulness of immunohistochemical markers to the diagnosis of early myocardial damage has been recently suggested because most of them can be visible even serologically as early as few minutes after the beginning of the symptoms. To evaluate the usefulness of plasma and cellular antigens, their distribution patterns have been studied among a group of 18 SCD cases in which a myocardial ischemia was strongly suspected. For the present study, 4 markers have been selected on the basis of their different diagnostic potential as follows: among the plasma markers the C5b-9 and fibronectin, among the cellular markers the myoglobin and cardiac troponin. The results show that only the study of multiple markers such as those selected can provide enough evidence of myocardial ischemia and/or necrosis, supporting the final diagnosis of SCD. No single immunohistochemical staining is ideal for diagnosing early myocardial ischemia but a set of markers can improve the ability of forensic pathologists to detect ischemic areas when no macroscopic or microscopic evidence of necrosis is available. However, the interpretation of data obtained in each individual cannot be isolated from the overall assessment of the factors (cardiopulmonary resuscitation and/or agonal artifacts) that can affect the expression of each marker.

Undetected patricide: Inaccuracy of cause of death determination without an autopsy.

Patricide (killing the father) is uncommon form of homicide. Usually the assaults occur at home in the absence of witnesses and adult sons are frequently involved. Homicides in a domestic context usually do not tend to recurrence, because the motivation for the crime ends with the death of the parent. However, this is not what was observed in the present case study dealing with the death of a 70 years old white man originally misclassified as accident and discovered three years later only after an additional homicide in a family context of a 60 years old white lady. Multiple stab wounds to the neck and thorax were misinterpreted at the external male body examination as blunt trauma falling down stairs. No forensic autopsy was requested and no comparison of medical findings with the results from the death scene, such as a bloodstain analysis was performed by the police officers nor required by the judicial authority. This was quite surprising because an additional but preliminary post-mortem external examination performed by a general practitioner on the male body already raised the suspicion that the external lesions were stab wounds thus requiring a forensic autopsy. Only the exhumation of the elderly body, performed years later, confirmed the diagnostic hypothesis raised by the first physician. The present case is quite representative of a death investigation not run professionally and performed by individuals with no specific training where most of the medico-legal investigations (especially for traumatic and violent deaths) are restricted to an external body examination without subsequent autopsy. Although misinterpretation of external lesions is inevitable and significant discrepancies between external body examination and forensic autopsy are not rare, in the case of contradictory results of post-mortem external examination or unclear/suspicious cause and manner of death, investigation should proceed necessarily with a forensic autopsy.

Investigation of Italy's deadliest building collapse: forensic aspects of a mass disaster.

We describe the investigation of the 1999 collapse of an apartment building in Foggia, Italy. Sixty-one victims were recovered in the rubble of the building, and five people were unaccounted for. All the bodies were well preserved except for two who had been burned. The majority of the victims were identified visually or by comparing body features, clothing, or personal effects with information collected from relatives or friends. Positive identifications of the two victims who were burned were obtained by dental comparison and DNA analysis. Approximately half of the victims (51.6%) sustained fatal injuires, while the remainder died from asphyxia. The injuries were characterized using the Abbreviated Injury Scale (AIS) and the New Injury Severity Score (NISS) systems. Injury severity associated with the location of victims inside the apartment may provide useful information for those involved in building design and/or search and rescue operations. Engineers determined that the collapse was the result of the use of inappropriate foundation material.

The bodies of two missing children in an enclosed underground environment.

Two young children, respectively 11 and 13 years old, originally from a small town of Southern Italy, were missing on 5 June 2006 (06:30 p.m.). Soon after their disappearance, the search for the missing children began. Broadcasters promptly transmitted a description of the missing children, pushing the entire community to assist in the search and safe recovery of the children. However, every effort was in vain, and the search went on for more than 1 year. During the missing-child search, the investigators collected enough evidence against the father, who was arrested 17 months after the children's disappearance. He was indicted for kidnapping, homicide and concealment of the two bodies. He never confessed to the crimes and claimed to be innocent. Three months after the conviction, a fireman found the two corpses in a subterranean, dry cistern next to a well over 20 m deep. The bodies were well preserved, almost mummified, with only few body-parts skeletonised. Based on dental records, they were identified as those of the two children, who had gone missing 1.5 years before. Signs of a very low insect activity were present, reasonably consistent with a rapid skin dehydration. The autopsy showed no signs of defence injuries or ligature consistent with strangulation or captivation, except for fractures of the axial skeleton at a number of points consistent with a fall from a low-medium height. The body of the elder brother presented major injuries with signs of recent haemorrhages and gut content analysis consistent with the last meal, which provided enough evidence to ascertain a very short survival time. The younger child showed minor injuries, signs of old haemorrhages and gastric- and intestine-emptying time consistent with a longer survival time of approximately 3-4 days, spent alone in the dark and cold cistern. A long post-mortem interval (PMI) of approximately 20 months was estimated, mainly from the pattern of insect succession. Based on such physical evidence, on 4 March 2008, 4 months after conviction and 40 days after the recovery of the two bodies, the father was finally released from prison and exonerated from previous indictment of homicide.

DNA degradation and genetic analysis of empty puparia: genetic identification limits in forensic entomology.

Puparial cases are common remnants of necrophagous flies in crime investigations. They usually represent the longest developmental time and, therefore, they can be very useful for the estimation of the post-mortem interval (PMI). However, before any PMI estimate, it is crucial to identify the species of fly eclosed from each puparium associated with the corpse. Morphological characteristics of the puparium are often distinctive enough to permit a species identification. But, even an accurate morphological analysis of empty puparia cannot discriminate among different species of closely related flies. Furthermore, morphological identification may be impossible if the fly puparia are poorly preserved or in fragments. This study explores the applicability of biomolecular techniques on empty puparia and their fragments for identification purposes. A total of 63 empty puparia of necrophagous Diptera resulting from forensic casework were examined. Samples were divided into three groups according to size, type and time of eclosion in order to verify whether the physical characteristics and puparia weathering can influence the amount of DNA extraction. The results suggest that a reliable genetic identification of forensically important flies may also be performed from empty puparia and/or their fragments. However, DNA degradation can deeply compromise the genetic analysis since the older the fly puparia, the smaller are the amplified fragments.

Cross-cultural comparison of two serial sexual murder series in Italy and the United States.

There have been few documented comparisons of serial murder cases committed in the United States with cases occurring internationally. The authors contrasted two unique serial murder series: one in Italy and one in the United States by examining the details of both series, including the M.O., motivation, crime scene interactions, sexual acts performed, and the general backgrounds of both offenders. The comparison revealed a number of similarities. Both offenders specifically targeted elderly women, who were attacked in their residences. The two series involved sexually motivated crimes, although the sexual interactions were different. Both offenders stole objects from their victims after the murders and each kept newspaper accounts of their crimes. In addition, both offenders claimed to have abusive upbringings, including sexual abuse.

A serial killer of elderly women: analysis of a multi-victim homicide investigation.

Between 1995 and 1997, in the territories of Southern Italy, there were fifteen murders of elderly women over the age of 70 years old. Initially, however, not all the murders were attributed to a single serial killer. The majority of the victims were stabbed multiple times in the neck, except for three cases in which the cause of death was manual strangulation. There was evidence of sexual assault in only one of the cases. All the victims were discovered in their own apartments, which were located on the ground level, with no signs of forced entry. In most of the cases, the offender stole money and/or jewellery. A multi-disciplinary team reviewed the cases during the investigation and created a profile of the killer. The team determined that the method of operation was completely unusual for the local criminal element. They suggested that the perpetrator could be an immigrant, who committed the murders for sexual motivation and who may have been arrested previously for sex-related incidents. On 15th September 1997, a suspect was arrested. He was identified as Ben Mohamed Ezzedine Sebai, a 35-year-old white male, originally from Tunisia. He was charged and convicted of four of the murders and was given a life sentence. In 2005, Sebai confessed to the murders of four additional elderly women, for which nine other people had already been previously tried and convicted, among them, a man who committed suicide in jail. In 2007, Sebai finally confessed to committing fifteen murders that occurred between 1995 and 1997. Sebai also admitted to experiencing sexual gratification at every homicide scene, even though there was no physical proof at most of the crime scenes. The goal of this article is to illustrate a little-known but noteworthy case concerning a serial sexual killer of elderly women that occurred in Southern Italy, highlighting the method of operation, the victim selection process, and the injuries inflicted. The article will also discuss his motivation, the mental health history of the offender, his clinical diagnosis, and his self-reported childhood abuse.

A promising microbiological test for the diagnosis of drowning.

A number of biological and chemical tests have been developed over the years to determine whether a person was drowned. This study focuses on the potential of a microbiological test for detecting common bacterial markers of water faecal pollution such as faecal coliforms (FC) and faecal streptococci (FS) as possible indicators of drowning. A promising previous study was carried out on central and peripheral blood samples of 42 drowned victims (20 cases in saltwater and 22 cases in freshwater) and 30 not-drowned bodies. To improve the accuracy of our previous results and also in order to investigate a possible cause of a false positive due to pulmonary passive diffusion and subsequently endogenous or exogenous bacterial invasion of the blood in the post-mortem interval (PMI), the FC and FS test was applied to bodies submerged in water but died from causes other than drowning. In the present study, blood samples collected from the left ventricle (LV), right ventricle (RV), femoral artery (FA) and, femoral vein (FV) of 10 drowned victims (5 cases in freshwater and 5 cases in seawater) and 3 not-drowned individuals with bodies submerged in water for a while after death have been analysed. Preliminary results are in agreement with other reports dealing with diatoms and marine bacteria that suggest to exclude the hypothesis of a passive penetration of sufficient quantities of drowning medium into circulation after death or during the agonal period. Based on our results there is also no evidence of a relevant dissemination of endogenous micro-flora from the gastrointestinal tract affecting the FS and FC test. There are still several other factors that could influence the applicability of post-mortem FS and FC cultures for the diagnosis of drowning and they need further investigations. The present article provides only a glimpse of the potential of the FS and FC test as bacteriological method for the diagnosis of drowning.

Craniofacial identification by comparison of antemortem and postmortem radiographs: two case reports dealing with burnt bodies.

Two cases of positive identification of burnt bodies by radiographic comparison are reported. They emphasize that antemortem radiographs of the head are an important but sometimes overlooked source of information which can frequently provide useful objective data for comparison purposes. A positive identification can easily be achieved by medical examiners through visual comparison of the antemortem with the postmortem cranial and facial structures, even of bodies severely damaged by fire. In these bodies the radiographs of the skull can graphically depict structures which are often unique to the individual, such as the frontal sinus pattern and the morphology of dental restorations. However, the process of identification through radiographs is appropriate only in burnt bodies in which antemortem radiographs of the alleged deceased are available for comparison and unique craniofacial structures are still present on the body in a well-preserved state or at least not completely destroyed by fire, depending on the extent of the burn injury. Matching of corresponding features seems preferable to other methods of personal identification such as skull-photo superimposition, morphometric analysis, and/or other computer-aided methods since these techniques need trained personnel, as well as expensive equipment which is not invariably available in the medical examiner's office or department of anthropology.

Best practice in forensic entomology--standards and guidelines.

Forensic entomology, the use of insects and other arthropods in forensic investigations, is becoming increasingly more important in such investigations. To ensure its optimal use by a diverse group of professionals including pathologists, entomologists and police officers, a common frame of guidelines and standards is essential. Therefore, the European Association for Forensic Entomology has developed a protocol document for best practice in forensic entomology, which includes an overview of equipment used for collection of entomological evidence and a detailed description of the methods applied. Together with the definitions of key terms and a short introduction to the most important methods for the estimation of the minimum postmortem interval, the present paper aims to encourage a high level of competency in the field of forensic entomology.

Pink teeth in a series of bodies recovered from a single shipwreck.

Pink teeth have most often been observed in victims of drowning but have also been reported in subjects who died suddenly and unnaturally. There is general agreement that there is no obvious connection between the occurrence of pink teeth and the cause of death, but the condition of the surroundings (especially humidity) must certainly play an important role in the development of the pink-tooth phenomenon. The frequency and distribution of postmortem pink coloration of the teeth have been studied among a representative sample of 52 cadavers. All the bodies were victims of a single shipwreck that occurred on March 13, 1997, in the middle of the Otranto Canal (Mediterranean Sea). The bodies were recovered from the seawater after approximately 7 months. A distinct pink coloration of the teeth was found in only 18 cadavers (13 females and 5 males) of ages ranging between 13 and 60 years. The phenomenon was more pronounced in younger individuals due to age-related changes of the root canal, less penetrable by the pigment responsible for the postmortem pink staining. By histochemical methods and autofluorescence, hemoglobin and its derivatives have been identified as the most likely pigments responsible for this postmortem process that can be considered analogous to postmortem lividity. These data are consistent with previous reports on pink teeth, indicating that the diffusion of the blood in the pulp into the dentinal tubules causes the red discoloration of the teeth. Based on the results, the pigmentation is more prominent on the teeth with single roots rather than in the posterior teeth with multiple roots.

Forensic genetic analysis of insect gut contents.

Entomological evidence is most often used for estimating the postmortem interval, but fly larvae can also be a source of vertebrate DNA. Forensic analysis of DNA recovered from a larva's gut can be used to identify what the larva had been feeding on. During our previous research studies, we used the same DNA extraction for the dual purpose of identifying the insect species and associating a maggot with its last meal. In our experience, we have encountered several situations where this method for associating a maggot with a corpse would have been useful, such as removal of remains from a suspected crime scene, an alternative food source is nearby the scene or the body, and a chain-of-evidence dispute. However, since maggot gut content analysis is a quite brand-new area of study, many of the limitations of the technique have not yet been explored. The results of our most recent research studies suggest that third-instar larvae actively feeding on the corpse can be considered the best source of human DNA, better than postfeeding or starved larvae. In this paper, the state of the art of forensic genetic analysis of maggot gut contents is reviewed.