Abstract
The authors used a particular protocol on putrefied corpses to highlight the cutaneous furrow. Two groups of cadavers were selected: in the first group (suicide by hanging mechanical asphyxia), the authors sampled cutaneous lozenges on the furrow still macroscopically recognisable, while in the second group (corpses deceased by different means), we sampled cutaneous lozenges in the hypostatic leaning. All specimens were divided in two: one underwent standard fixation while the other, previously rehydrated in Sandison’s solution, was then fixed in formalin. All specimens were stained with hematoxylin and eosin and Resorcin-Fuchsin. Samples processed with formalin and the H&E staining underlined various artifacts; vice versa, the Sandison solution associated with Resorcin-Fuchsin staining demonstrated the compression of the elastic fibres, with focal positivity in hanged corpses, and diffuse positivity in the entire hypostatic leaning. Sandison’s rehydrating solution and Resorcin-Fuchsin staining exalt the supposed application of an asphyctic mean even on putrefied corpses in those cases burdened by a diagnostic doubt.
Introduction
The characteristic depression of the cutaneous surface of the furrow produced during hanging is usually recognisable in well-preserved corpses but putrefaction and post-mortem loss of liquids acting on already injured cutaneous areas can cause macroscopic artifacts concealing the furrow or mimicking fictional traumatic lesions,1–3 creating a pronounced thickening of the specimens and various technical artifacts during microscopic observation. For medico-legal purposes, further assistance using laboratory techniques is therefore needed for a reliable diagnosis. On cadavers with dubious hanging lesions, which are not clearly macroscopically detectable due to advanced autolytic processes, the microscopic characteristics normally used to identify the furrow – compression and fragmentation of the superficial epithelium, compression of dermis-hypodermis, intra-vitam haemorrhagic infiltration – can be altered or missing due to detachment/loss of the epithelium, oedema, fragmentation and degeneration of the dermis and red blood cells autolysis. The elastic tissue is the only structure which is resistant to putrefactive phenomena, even when this is advanced, and which can be rapidly and economically and satisfactorily demonstrated with histochemical techniques. 3
Using the marked resistance to decay of the elastic tissue as a starting point, we investigated possible structural alterations of the elastic fibres in two groups of cadavers which were extensively decayed and putrifying. In the first group, the victims died from hanging and mechanical asphyxia: the study focused on the compressed skin caused by the noose; in the second group of cadavers who died from causes other than from hanging, our attention focused on the leaning regions of the cadaver to evaluate possible compressive effects caused by the victim's weight. The purpose of this study was to evaluate the application of the Resorcin-Fuchsin staining, specific for elastic fibres to demonstrate any possible alterations caused by compression and to assist reaching a diagnosis in cases of hanging which had caused mechanical asphyxia where the body was putrified or badly decayed when discovered and first examined.
Materials and methods
The study was carried out at the Institute of Legal Medicine of the University of Milan from July 2015 to July 2016 on cadavers undergoing judicial autopsy on behalf of the Inquiring Magistrate: three victims (Group 1) apparently dead from mechanical asphyxia due to hanging were discovered when advanced putrefaction had occurred. The dermis of the cervical region in the context of the furrow was sampled and examined.
The control group (Group 2) had the same number of bodies but these deceased had died from different causes (myocardial infarction, natural intra-parenchymal cerebral haemorrhage and acute narcotism). Samples of the dermis were taken from areas of the body that had been compressed due to the position of the body (dorsal and gluteus regions, knees), to compare any structural alterations of these elastic fibres with those resulting from the weight of the body when left hanging, analysing the depth in the dermis and the longitudinal involvement across the samples.
Six cutaneous lozenges were sampled (0.5 mm × 2.0 cm × 2.0 cm) from the cutaneous furrow and from different cutaneous regions in the control-corpses: these samples were divided into two similar halves. Half were directly fixed in 10% buffered formalin, while the other half were processed with Sandison’s rehydrating solution 4 for 48 h and then fixed in 10% buffered formalin for 48 h. Once these phases were concluded, the cutaneous fragments were processed with classical dehydration in absolute ethyl alcohol, clarified in a substitute of xylene and impregnated in paraffin. Sandison’s rehydrating solution was used as it allows for easier preparation of cutaneous samples stiffened by post-mortem liquid loss, 5 by compression resulting from hanging or by compression from the weight of the body on tissue in contact with the ground.
Two 3 µm thick slides were prepared for each case, using a sledge microtome, then stained with hematoxylin eosin (H&E) and Resorcin-Fuchsin; the latter is specific for elastic fibres. In the Resorcin-Fuchsin staining, the slides were not counterstained with the van Gieson’s technique in order to avoid the treatment with picric acid that could have caused a loss of reactivity and a fainter reaction of the elastic fibres. 6 The 24 slides obtained were then examined using a Leica DMR microscope and the most significant images were captured with a Leica DC300F digital camera.
Cases presentation – Group 1
Case 1
The residents of an apartment block were worried by a persistent and strong odour coming from the apartment of a 51-year-old man who had not been seen for a week and called the police who entered the apartment where they found his hanged body with a farewell note which stated that his suicide was a consequence of his recent divorce from his wife. The Judicial Authority sent a request for an autopsy to the Institute of Legal Medicine of the University of Milan three days later.
The cadaver (height 180 cm, weight 66 kg) was in an emphysematous/liquefactive phase and colonised by larvae. The neck was wrapped in a greyish rope 1 cm wide, wound over the larynx and fixed with a metallic snap-hook. Once the noose was removed, a whitish cutaneous furrow with oblique anterior-posterior direction was revealed: its maximum depth (1 cm) and width (3.5 cm) were in the frontal median area. No other significant evidence was collected during external examination (Figure 1(a)). During cervical dissection, the pathologist highlighted a diffuse orange-brownish homogenisation of the muscular-fascial structures over and below the hyoid bone; the hyoid bone and the superior cornus of the thyroid cartilage were found to be fractured and showed haemorrhagic infiltration. No other significant evidence resulted from the internal examination and the cause of death was identified in mechanical asphyxia by hanging.
(a) Macroscopic side overview of the cadaver with the rope still in place. (b) detailed overview of the ligature and of the cutaneous furrow.
Case 2
The landlord of an apartment, seeking to collect rent which was several months overdue from his 53-year-old tenant, discovered his hanged body and informed the police. The Judicial Authority ordered the autopsy at the Institute of Legal Medicine of Milan a week later. In the flat, during crime scene investigation, the pathologist found a farewell note which explained he had committed suicide because of money problems caused by the recent loss of his job.
The corpse (height 178 cm, weight 35.6 kg) was in a liquefactive phase and was extensively colonised by larvae. A thin plastic rope, with a single knot in the right paramedian region of the nape, was round his neck. Once the noose was removed, the forensic practitioner found a whitish cutaneous furrow, flat, with an oblique anterior-posterior progression and a circumference of 34 cm. No other significant evidence was collected during external examination of the corpse (Figure 1(b)). During cervical dissection, the pathologist highlighted a diffuse brown-greenish coloration of the underlining layers; there were no relevant findings at the dissection of the anterior muscular cervical layers, the hyoid bone was intact, the superior cornus of the thyroid was found to be fractured and showed a haemorrhagic infiltration of the surrounding soft tissues. The cause of death was stated as due to mechanical asphyxia caused by hanging.
Case 3
A 55-year-old man was found by dustmen who had just started their shift; he was hanging from a tree branch in a park in the northern part of the city of Milan. The Judicial Authority ordered the post-mortem examination at the Institute of Legal Medicine of the University of Milan four days later. The victim's relatives explained that the deceased had suffered from major depression for a long time, and that he had recently been evicted from his home and had lost his job. The deceased had never sought medical assistance.
The corpse (height 170 cm, weight 70 kg) was in an emphysematous phase and was positioned on the dissection table with the rope still round his neck, with a single coil and a single knot in the right mastoid region. Once the rope was removed, a bruised-excoriated furrow was observable in the super-laryngeal region, strip-shaped, red-yellowish, running in an oblique downward-upward direction from left to right, with regular edges and rough bottom, splitting into two branches in the lateral-cervical region. The maximum height was 2.1 cm and the maximum depth of the lesion was 0.4 cm in the left anterior paramedian region, showing haemorrhagic crests and serum-filled blisters. No other relevant finding could be noted at the external examination of the cadaver. During cervical dissection, the pathologist noticed a haemorrhagic infiltration of the lower third of the right sternocleidomastoid muscle and haemorrhagic infiltration of the left soft tissues around the lesion in the context of the cutaneous furrow. No other significant finding was noticed and the cause of death was identified as hanging mechanical asphyxia.
Cases presentation – Group 2
The victims of this group were selected according to three parameters that we considered relevant for this study: the cause of death had to be different from hanging mechanical asphyxia, the putrefactive phenomena had to be advanced and the victim had been lying against a flat surface with at least part of his body.
The first case, a 62-year-old heart patient (height 176 cm, weight 67 kg), was discovered lying on his back on his bathroom floor in an emphysematous phase. The post-mortem examination identified the cause of death as myocardial infarction in a person with severe coronary artery disease. The most compressed area caused by the weight of the body was the dorsal left scapular region.
The second case, a 54-year-old man suffering from hypertension (height 172 cm, weight 71 kg), was being treated with hypertensive drugs and was discovered in his car in a emphysematous/liquefactive phase. The cause of death was identified as natural cerebral intra-parenchymal haemorrhage. The cutaneous regions most subjected to compression were the gluteus areas, on both sides.
The third case, a 39-year-old, (HIV) HCV positive male drug addict (height 174 cm, weight 71 kg), was discovered kneeling on the floor of his kitchen, in a emphysematous phase. The cause of death was identified, after post-mortem examination and toxicological analysis, as a drug overdose. The cutaneous regions most affected by compression by his body weight were his knees.
Results
Samples fixed in formalin
In both groups of cadavers, H&E staining demonstrated effects that could be traced to the degradation of tissues: homogeneous degradation and loss of the epidermic surface, rough putrefactive bacterial colonies, marked eosinophilia of the dermic region, hair follicles partially recognisable; the adipose sub-dermic tissue appeared to be still recognisable, while the muscular tissue had dissociated fibres, hyper-eosinophilic cytoplasm and pyknotic nuclei, when still present. In both groups, the Resorcin-Fuchsin staining confirmed perfect conservation of the elastic tissue, in the dermis and in the wall of the vases. In all the Group 1 cases, in the area of the furrow, elastic fibres appeared focally compressed and strongly condensed in the papillary dermis, especially in the sub-epithelial region and in the reticular dermis (Figure 2(a) to (c)); in the areas surrounding the furrow, condensation of these fibres appeared modest. In Group 2, the samples from case no. 1 (Figure 2(d)) and 3 (Figure 2(e)) demonstrated elastic fibres diffusely compressed across the entire sample with minimal differences that could be correlated to the weight pressing against the surface on which the corpse was lying, while in case no. 2 (Figure 2(f)), the elastic fibres had normal structure and disposition.
Samples fixed in formalin and stained with Resorcin-Fuchsin. a (100×), b and c (200×), portions of the cutaneous furrow with pronounced compressive effect of the elastic structures of the profound reticular dermis. d (100×), lying region (dorsal area) with compressive thickening of the papillary dermis across the entire sample. e (100×), lying area (knee) with widespread compressive and dehydrated effects of the elastic fibres, homogeneous across the sample. f (200×), lying area (gluteus region) with normal architecture of the elastic fibres in reticular and papillary dermis.
Samples previously processed with Sandison’s rehydrating solution and then fixed in formalin
Both groups showed at the H&E staining microscopic similar changes to those observed in the samples directly fixed in formalin. The dermis, however, appeared to be more compact, homogeneous and faintly hyper-eosinophilic. The histochemical staining with Resorcin-Fuchsin, in Group 1, demonstrated focal compression and thickening of the elastic fibres in the reticular dermis in the context of the cutaneous furrow while, more superficially, in the papillary dermis, the rehydration accomplished with the Sandison’s solution gave a milder picture of these aspects (Figure 3(a) to (c)). In the areas surrounding the lesion, the architecture of the elastic fibres appeared to be more regular without evident signs of compression. In Group 2, the processing with Sandison’s rehydrating solution did not produce substantial improvements to the samples analysed and stained with Resorcin-Fuchsin, showing a constant aspect and distribution of the elastic fibres in the specimens (Figure 3(d) to (f)).
Samples processed with Sandison’s solution, fixed in formalin and stained with Resorcin-Fuchsin. a (100×), b and c (200×), samples of furrow showing compression of the elastic fibres. d (100×), lying region (dorsal area) with areas of elastic fibres thickening in the sub-epithelial papillary dermis. e (100×), lying area (knee) with evident effects of elastic fibres compression. f (100×) lying region (gluteus area) with normal architecture of the elastic fibres of the reticular and papillary dermis.
Discussion
Histochemical techniques and laboratory analysis are very useful and often fundamental in medico-legal investigations, enabling a confident diagnosis to be reached especially when there is no macroscopic evidence.
When the forensic practitioner examines a corpse undergoing extensive putrefactive phenomena, the diagnosis of a constrictive cause of death can be very difficult because the detrimental mean leaves undefinite mascroscopic signs. Microscopic evaluation of histological samples is unreliable. We examined the skin of three decaying corpses of people who had died from hanging with the means used still in place (Group 1) to evaluate possible architectonical alterations of the cutaneous elastic fibres, known to be more resistant to decomposition around the cutaneous furrow. These three cases were compared to three controls (Group 2) where the deceased had died from other causes than hanging asphyxia but where the bodies had decayed and their weight was resting on different areas of the body: dorsal or gluteus regions or knees and a skin sample were taken to evaluate the state and morphology of the elastic fibres.
To improve the preparation of these samples and facilitate the histological analysis, we combined the classical fixing with 10% buffered formalin and the processing with Sandison’s rehydrating solution followed by classical 10% buffered formalin fixation since, according to literature on highly degraded specimens,4,5 this procedure improves the histochemical structure, especially skin. In order to highlight elastic fibres, we stained them with Resorcin-Fuchsin dying without counterstaining according to van Gieson’s technique to avoid its principal component, picric acid, discolouring these delicate fibres already damaged by autolytic processes.6,7
In both series of cases (fixed with formalin and rehydrated with Sandison’s solution), we noticed an obvious alteration with thickening of the elastic fibres in the papillary and reticular dermis. The compressive effect was more evident in the samples processed with formalin, while the Sandison’s rehydrated specimens showed a still marked compression in the reticular dermis but less pronounced thickening in the papillary dermis, probably caused by the greater rehydration in the superficial area. The extension of the thickening along the sample demonstrated that the abnormal effect of compression of the elastic fibres had a different pattern in the two groups: in the samples belonging to Group 1, elastic fibres appeared to be compressed only focally in the context of the cutaneous furrow and having a normal architecture in the nearby regions. The disposition of elastic fibres in the passage areas (cutaneous furrow-normal skin) appeared to be neat, with a flattened aspect in the injured area and normal aspect in the healthy regions. In Group 2, elastic fibre samples were in two cases out of three more regular, with generalised compression along the entire sample; the architecture of the elastic fibre in the passage areas (lying compressed skin–not compressed skin) appeared to be blurred, with a progression to normality. The lack of elastic fibres’ compression in case no. 2 (victim discovered sitting in his own car) was attributed to the leather seats that spread the surface pressure on the body.
The study of histochemical techniques on the alteration of the elastic fibres due to compression in cadavers which are decayed and putrifying, of people who died from hanging, therefore provided interesting, albeit preliminary, results. The combined application of Sandison’s rehydrating solution and the Resorcin-Fuchsin staining should be considered useful in all cases where routine laboratory procedures are insufficient to produce a confident diagnostic hypothesis on the cause of death in victims of mechanical asphyxia due to hanging.
Footnotes
Authors’ contribution
All the authors listed contributed equally to this work.
Ethical approval
This article does not contain any studies with living human participants performed by any of the Authors. All the studies conducted followed the guidelines provided by Legislation and the National Bioethical Committee.
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.