When investigating a violent crime, one crucial question is under what circumstances the victim died. To this end, the external examination of the body is mandatory and can provide key information. Several types of materials or traces can be collected from a corpse: for example biological of human origin (i.e. blood, sperm, saliva), biological of nonhuman origin (i.e. traces of botanical, fungal, or animal origin), inorganic (i.e. varnishes, gunshot residues) and traces of soil or minerals. Although the analysis of all these trace materials collected on the body may provide circumstantial evidence(s) [
1,
2], not all of them receive equal consideration in the medicolegal context. Those of human origin are one of the main objects of interest and are universally included and standardised in all protocols of approach to the body (at least in the case of well-preserved bodies); instead, the others are often neglected, and their management is left to the discernment of the individual operator [
3‐
5]. Nevertheless, the fact that the environment leaves macroscopically visible or invisible traces on the victim is not in dispute [
6,
7], and, in some cases, the macroscopically invisible trace could be the only trace of the environment where a crime occurred. The need arose from homicide cases that the authors dealt with in recent years, the most significant of which concerns the partially skeletonized corpse of a girl found in a field. In that case, several elements suggested that the victim may have been killed elsewhere and subsequently deposited where the discovery occurred. For this purpose, several investigations aimed at studying the interaction between the corpse and the environment were carried out. Skin and clothing samples were taken upon autopsy examination and subjected to SEM/EDX analysis. Samples revealed the widespread presence of micro-traces rich in calcium powders (calcium oxide, CaO) and numerous metallic spheres made of iron (Fe), chromium (Cr), and/ or nickel (Ni). The source of such material was sought in all the environments usually visited by the victim without finding matches. So, a forensic geologist and a mechanical engineer were requested. They direct the research toward an environment connected with the construction industry; this element allowed the identification of the main suspect for the homicide [
8]:
Building on this experience, the present work aims at reconstructing the interaction between a cadaver and six different environments simulating a hypothetic crime scene: skin samples were placed in five different workplaces and inside the trunk of a car. The samples were then studied using four methods of investigation: a morphological one with the naked eye, with an episcopic microscope, and through Scanning Electron Microscopy (SEM) and a spectroscopic approach by Energy-dispersive X-ray spectroscopy (EDX) and Energy Dispersive X-Ray Fluorescence (ED-XRF). Both SEM–EDX and ED-XRF are well-known techniques for the search of information for the analysis of GSR (gunshot residues) on both skin and garments [
5,
9‐
14] or to identify the material of a weapon [
15‐
19], but rarely used to pinpoint a victim's environment of origin or passage.
Hence the goal of the present study is to verify, as done previously for several inorganic substrates [
20‐
25], if and how skin could be the recipient of traces from the environment and highlight the importance of corpse debris research to identify where the body has been exposed or what it has been put in direct contact with and to evaluate any qualitative changes of the debris over time, with particular attention to the progression of putrefactive phenomena.
Materials and Methods
A swine skin was designated as the optimal source for producing the samples, due to its similarity with human skin [
26]. The skin was obtained from an animal that died from causes unrelated to this study and, immediately after recovery, was stored in a freezer at -20 °C. Half of its surface was shaved. The entire skin was thoroughly rinsed with water and then cut in 122 squares measuring 3 × 3 cm each.
Six sites were selected for experiments reported in this paper: a florist, a bakery, a carpentry, a turnery, a trunk of a car, and a construction site. The following samples were placed in each experimental environment:
-
Two squares, one hairy (H +) and other one hairless (H-), were put in direct contact with a plane surface of the environment (floor or tables, shelves) by adhering the upper surface of the skin sample for 30 s.
-
Two squares, one hairy (H +) and other one hairless (H-), were exposed to the open air in the same environments for 4 h; in this case, the sample was placed on a plane with the upper surface exposed to the air.
The following control samples were provided:
-
One hairy square (H +) and one hairless (H-) underwent the same analysis intended for studying the other samples without being exposed to the environment.
-
One graphite stub (suitable for direct analysis by SEM–EDX because it is electrically conductive) was exposed to the environment for 4 h and one put in direct contact with the same plane surface selected for the test samples, for 30 s, and then analyzed by SEM–EDX.
Therefore, the number of squares was 4 for each of the six experimental environments and for each of the five experimental times, given the destructive nature of some techniques. The first set of 24 samples was studied immediately after the 4 h of exposure and after the direct contact with the environment (Time 0); the remaining samples were stored in plastic boxes open but protected from light with paper covers in a controlled environment. New sample analyses were carried out on the stored samples after 1 day (Time 1), after 1 week (Time 2), after 2 weeks (Time 3), and after 1 month (Time 4). Total skin samples studied was 122 [(4 × 6 (experimental environment) × 5 (experimental time)) + 2 (negative control samples)].
Each square, comprising the two negative controls, underwent four subsequent analytical processes:
-
naked eye observation
-
episcopic microscope observation (Leica Wild Heerbrugg Op Mikroskop M650)
-
double direct ED-XRF study on the upper half (Assing Lithos System)
-
SEM–EDX study of the lower half after application and graphite metallization of two 0.5 cm.2 graphite tapes (Cambridge Stereoscan 360)
The operating parameters and analytical methodologies applied with the different instruments are the following:
Naked eye—All samples were observed without instrumentation, photographed, and all material of interest was classified and recorded.
Episcopic microscope—Each sample was visually traversed twice following parallel lines at 16X and 25X magnifications from top to bottom. All material of interest was documented photographically and classified during this observation.
ED-XRF—The instrument was equipped with a low power X-ray tube with Mo anode and a zirconium transmission filter (100 µm) producing a monochromatic excitation spectrum. The working conditions were 25 kV and 0.3 mA. All samples were subjected to two analyses. Each analysis generates a graph showing the peaks corresponding to the chemical elements most represented in the investigated area, which corresponds to a surface of about 0.5 cm2 of skin.
SEM–EDX—The instrument has been equipped with lanthanum LaB6 processed filament, the acceleration voltage is set at 20 le and the working distance at 25 mm. Each sample is entirely explored at magnifications of 500X for a general overview and then specific elements were analyzed with magnifications up to 300.000X. All material of interest is documented photographically and through spectroscopic analysis, counted, and classified during this observation.
All the elements found with the different techniques in the environmental positive control samples (graphite stubs) of the six environments tested were assigned a value from 0 to 5, this score was produced by subtracting from 6 (total number of environments) a point for each environment that shares this element. This value represents the specificity score of these elements: score 0 if the element is present in every environment, and therefore useless for identifying or excluding any of them, score 1 if the element is present in 5 environments, so only valuable for excluding 1 of the 6, score 2 if the element is present in 4 environments, so only valuable for excluding 2 of the 6, score 3 if the element is present in 3 environments, and, with the same principle, score 4 if present in 2 environments and 5 if present in only in 1 environment. Five represents the maximum score and identifies elements exclusive to a single environment and therefore highly identifying. In this way, each environment's score was calculated for both morphological and chemical elements; a higher total score will therefore be given to environments (through the score obtained from the graphite samples used as positive control) with a greater number of elements and with exclusive elements. Similarly, skin samples were scored. In this way, it was possible to assess a score for each sample (denoted as
value) and then compare that score with those obtained by the control sample from the same environment (denoted as
tot). With the same criterion, scores were attributed to elements not present in the control samples. These elements, not coming from the environment of origin, were considered as contaminants, and counted as negative (denoted as
contaminants). In this case, the rarity of the element, so their presence in one or a few different skin samples, was considered a negative factor because the more ubiquitous the contaminant is, the less misleading it will be for future interpretation [
27].