Carlo P. Campobasso

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.

Factors affecting decomposition and Diptera colonization

Understanding the process of corpse decomposition is basic to establishing the postmortem interval (PMI) in any death investigation even using insect evidence. The sequence of postmortem changes in soft tissues usually gives an idea of how long an individual has been dead. However, modification of the decomposition process can considerably alter the estimate of the time of death. A body after death is sometimes subject to depredation by various types of animals among which insects can have a predominant role in the breakdown of the corpse thus, accelerating the decomposition rate. The interference of the insect community in the decomposition process has been investigated by several experimental studies using animal models and very few contributions directly on cadavers. Several of the most frequent factors affecting PMI estimates such as temperature, burial depth and access of the body to insects are fully reviewed. On account of their activity and world wide distribution, Diptera are the insects of greatest forensic interest. The knowledge of factors inhibiting or favouring colonization and Diptera development is a necessary pre-requisite for estimating the PMI using entomological data.

Forensic entomology: applications and limitations.

Forensic entomology is the science of collecting and analysing insect evidence to aid in forensic investigations. Its main application is in the determination of the minimum time since death in cases of suspicious death, either by estimating the age of the oldest necrophagous insects that developed on the corpse, or by analysing the insect species composition on the corpse. In addition, toxicological and molecular examinations of these insects may help reveal the cause of death or even the identity of a victim, by associating a larva with its last meal, for example, in cases where insect evidence is left at a scene after human remains have been deliberately removed. Some fly species can develop not only on corpses but on living bodies too, causing myiasis. Analysis of larvae in such cases can demonstrate the period of neglect of humans or animals. Without the appropriate professional collection of insect evidence, an accurate and convincing presentation of such evidence in court will be hampered or even impossible. The present paper describes the principles and methods of forensic entomology and the optimal techniques for collecting insect evidence.

The forensic entomologist in the context of the forensic pathologist’s role

An adequate death investigation requires the combined efforts and cooperation of experts in different disciplines: crime scene technicians, death investigators, forensic pathologists, anthropologists, entomologists, other medical and non-medical professionals. These front-line experts play a crucial role in every death investigation process. The forensic pathologist normally has the legal authority to take charge of the dead body at a death scene and his primary functions are the exterior and interior examination of the cadaver by analyzing the extent of antemortem injuries and the postmortem changes and the recovery of physical evidence. He is responsible for determining how, when and why of any death which is the result of violence, suspicious or unexplained circumstances or a death which is sudden or unattended, defending and explaining the reasons for making these diagnoses in a courtroom. The forensic entomologist can provide invaluable aid in death cases where human remains are colonized by insects and in the overall investigation. His principal role is to identify the arthropods associated with such cases and to analyze entomological data for interpreting insect evidence. He is responsible for determining the period of insect activity according to all the variables affecting insect invasion of remains and their development. The major goal of medico-criminal entomology is to contribute to the determination of the time, cause, manner and place of the investigated death (especially on badly decomposed corpses or skeletonized human remains) with the support of all the elements which can be inferred from the study of insects found on the cadaver or nearby. The application of techniques devised recently in forensic entomology can allow experts in the field to collect strong entomological evidence and provide useful information not only in a death investigation including movement or storage of the remains following death, time of dismemberment, postmortem artifacts on the body but also at the scene, and even more in child neglect, sexual molestation and identification of suspects. As the role of the forensic entomologist at the death scene, at the autopsy and in the laboratory is defined and well known, this paper focuses on the difficulties that could arise if forensic pathologists and entomologists are uncertain about the procedures that they have to follow, do not realize the value of objective findings or fail to evaluate them. Although every forensic case presents a slightly different set of circumstances and has to be tackled individually, the forensic pathologist should work with the forensic entomologist from the visual observations of the cadaver on the scene, through the collection of arthropods and temperature data at the death scene and at the autopsy, up to the final report with the interpretation of entomological and other biological evidence.

THREE CASE STUDIES IN FORENSIC ENTOMOLOGY FROM SOUTHERN ITALY

Three cases of forensic interest regarding the estimation of postmortem interval (PMI) by entomological data are presented. The three cases concerning criminal investigations were performed in Southern Italy by the Entomological Laboratory of the Institute of Forensic Medicine at the University of Bari. For each case the authors present a detailed description of the remains as observed at the crime scene and a description of the arthropods collected from the remains. The PMI estimation was based on comparison of data from autopsy reports (rate of decay), local environmental conditions (temperature, humidity, rainfall) and development times for the immature stages of each species of local arthropod and succession patterns. The collection of insects was performed at the discovery site and during autopsy procedures. In the first case a PMI of 5 to 8 days was established based on the presence of adult specimens of Saprinus aeneus (family Histeridae), and mature larvae of Chrysomya albiceps and Sarcophaga carnaria (3rd instar). In the second case, on the charred remains of a corpse, larvae of Sarcophaga haemorrhoidalis (3rd instar) and Protophormia terraenovae (2nd instar) were observed in different developmental stages, as indicated, giving a PMI of 3 to 4 days based on entomological data. In the third case a PMI of 36 to 48 hours was defined from the evidence of Calliphora vicina 2nd instar on the two burnt bodies. In all cases the entomological evidence alone led to conclusions on PMI.

Sex determination by discriminant analysis of patella measurements

The authors have analyzed 80 skeletons (40 males and 40 females) from the collection at the Institute of Legal Medicine of the University of Bari belonging to a known contemporary Southern Italian population; time of death was around 1970 and ages ranged from 25 to 80 years. Seven measurements taken on 80 intact, undeformed right patellae (max height, max width, thickness, height and width of the external facies articularis, height and width of the internal facies articularis) were used to determine sex by multivariate discriminant analysis. One function associating two parameters (max width and thickness) obtained the highest value of correct sex determination with a rate of 83.3%; other functions showed a higher percentage of misclassification (up to 17.5%). This study tests the success rate of correct sex prediction based exclusively on patellar dimensions. The discriminant factors carried out by statistical analysis may aid the forensic anthropologist when no other human skeletal remains suitable for sex determination are available.

Sex determination by discriminant analysis of calcanei measurements

Eight measurements taken on the right calcaneus (maximum length, load arm width, minimum width, height of calcaneus, body height, breadth of the facies articularis talaris posterior, breadth and height of the facies articularis cuboidea) of a known contemporary Southern Italian skeletal population (40 males and 40 females) were used to determine sex by multivariate discriminant analysis. Three functions revealed a correct sex-determination of 85%. These functions were obtained by the association of the following parameters: maximum length, load arm width and breadth of the facies art. talaris post. (function no. 1); maximum length and breadth of the facies art. talaris post. (function no. 2); maximum length and height of the facies art. cuboidea and breadth of the facies art. cuboidea (function no. 3). These results may aid the forensic anthropologist when no other remains, useful for skeletal sex determination, are available.

Drug analysis in blowfly larvae and in human tissues: a comparative study

The present study investigates the correlation between concentrations of drugs in human tissues and Diptera larvae feeding on these tissues. Samples of liver were taken from 18 cases in which preliminary toxicological screening indicated the presence of drugs. Blowfly larvae (Diptera: Calliphoridae) were reared on these samples and subsequently analyzed for drug content. Toxicological analyses were carried out using ONLINE Abuscreen (Roche) and GC/MS for available body fluids (blood, urine and bile) as well as liver samples and maggots. All drugs detected in human tissues were also detected in insect specimens. Opiates, cocaine and barbiturates as well as some antidepressants (clomipramine, amitryptiline, nortryptiline, levomepromezine and tioridazine) were observed. Comparisons of drug concentrations between those in human tissues and blowfly larvae showed different patterns of distribution that may be attributed to differences in physiology. Results confirm the reliability of entomological specimens for qualitative analyses, although quantitative extrapolations are unreliable. All xenobiotics detected were in higher concentrations in human tissues than in maggots. Concentrations in post-feeding maggots were significantly lower than for feeding maggots, suggesting that the feeding state of maggots may affect toxicological analyses as they metabolize and eliminate drugs during development.

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.

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 worlds 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.