ORIGINAL ARTICLE
Diagnosis of Vitality in Skin Wounds in the Ligature
Marks Resulting From Suicide Hanging
Isabel Legaz Pérez, PhD,* Maria Falcón, PhD,* M. Gimenez, PhD,† F. Martínez Diaz, MD, PhD,*
M.D. Pérez-Cárceles, MD, PhD,* E. Osuna, MD, PhD,* D. Nuno-Vieira, MD, PhD,‡ and A. Luna, MD, PhD*
Abstract: Ascertaining the vital origin of skin wounds is one of the most
challenging problems in forensic pathology. The forensic literature de-
scribes biomarkers and methods for differentiating vital and postmortem
wounds, although no clear conclusions have been reached. The aim of this
study was to characterize human vital wounds by analyzing the concentra-
tions of metallic ions and the expression of P-selectin and cathepsin D in
skin wounds in the ligature marks in a cohort of suicidal hangings for
which vitality was previously demonstrated.
A total of 71 skin wounds were analyzed within a postmortem interval of
19 to 36 hours. The concentration of Fe, Zn, Mg, and Ca and the expression
of P-selectin and cathepsin D were analyzed together and separately. The
majority of autopsied suicidal hangings were men (86%) with complete
hanging mode (60.7%) in which there was a high frequency of subcutane-
ous injuries (78.3%). High concentrations of Ca and Mg compared with
Fe and Zn were found. Ca and Zn concentrations decreased, and Fe con-
centration increased with the seriousness of the injury. A high percentage
of moderately negative expression of both proteins was correlated with
subcutaneous injury and low or medium concentrations of Fe.
In conclusion, the joint study of metallic ions and proteins allows to char-
acterize and to differentiate an injured vital wound of noninjured skin, es-
pecially when the damage in the tissue affects to the majority of the
structures of the skin, but these results will need to be complemented with
other biomarkers in time-controlled samples to further help in the differen-
tiation of vital and postmortem wounds.
Key Words: cathepsin D, metallic ions, P-selectin, vital wounds,
suicidal hanging, forensic science
(Am J Forensic Med Pathol 2017;00: 00–00)
T he determination of skin wound age and demonstrating their
vital origin is one of the most central research areas in legal
medicine and an unresolved problem in forensic pathology. From
the point of view of legal medicine, it is often necessary to evalu-
ate the causal relationship between death and the wounds found on
the cadaver to discriminate antemortem wounds from postmortem
damage.1 Classically, it has been considered that the extravasations
of red blood cells and hemoglobin to the wound was a vital reac-
tion sign. However, several studies contradict these observations
Manuscript received September 18, 2016; accepted May 13, 2017.
From the *Department of Legal and Forensic Medicine, Biomedical Research
Institute, Regional Campus of International Excellence “Campus Mare
Nostrum,” Faculty of Medicine, University of Murcia, Murcia, Spain;
†Institute for Legal Medicine, Sao Paulo, Brazil; and ‡Institute for Legal
Medicine, Coimbra, Portugal.
The authors declare no conflict of interest.
This represents an original publication neither published nor under
consideration elsewhere. No funding was used to support this article.
Reprints: Isabel Legaz Pérez, PhD, Department of Legal and Forensic Medicine,
Biomedical Research Institute, Regional Campus of International
Excellence “Campus Mare Nostrum,” Faculty of Medicine, University of
Murcia, Campus de Espinardo, 30100 Espinardo, Murcia, Spain.
E-mail: isalegaz@um.es.
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ISSN: 0195-7910/17/0000–0000
DOI: 10.1097/PAF.0000000000000322
Am J Forensic Med Pathol • Volume 00, Number 00, Month 2017
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and conclude that neither can be used as a reliable marker in
wound vitality diagnosis.2
Several methods are described in the forensic literature for
the differential diagnosis of vital and postmortem wounds, includ-
ing analysis of the inflammation process,3–6 skin architecture,7–9
and cell adhesion molecules.10,11 In addition, when the behavior
of nucleic acids was studied, nuclear DNA degradation fragments
were observed.12–15 However, the use of specific vitality bio-
markers in an appropriate and standardized method is still a matter
of controversy for clarifying the relationship between wounds and
the cause of death.1,3,16,17
Selectins are adhesion molecules that have been found useful
to improve wound age estimation, especially in injuries with a
short survival time, whereas they are also indicative of the vital or-
igin of the wounds.18,19 Selectins are also carbohydrate-binding
molecules that bind to fucosylated and sialylated glycoprotein li-
gands, and are involved in the trafficking of innate immune sys-
tem cells, T lymphocytes, and platelets from blood vessels to the
tissue.20,21 More specifically, P-selectin (CD62P) has been found
in thrombocyte α-granules and in Wiebel-Palade bodies in the ac-
tivated vascular endothelium22 and have been studied for the dif-
ferential diagnosis of vital and postmortem wounds.10,23
Enzymatic markers have also been extensively studied as
wound vitality markers.24 Among them is cathepsin D, a lyso-
somal enzyme that plays an important role in certain apoptotic
processes, protein turnover, and protein processing.25,26 The en-
zyme is activated at the site of wounding to digest dead cells in
the low pH environment induced by hypoxia and necrosis. Ca-
thepsin D is the most widely expressed cathepsin in human skin,
and, although it seems to have features that make it a useful vitality
marker of potential forensic interest, past research has provided
no definitive and clear picture in this respect,27–29 because it
has been found to be highly expressed in both vital and postmor-
tem injuries.27 In addition, the differential diagnostic values of
metallic ions (Ca, Fe, Mg, Zn) have long been investigated as
possible markers for differentiating antemortem from postmor-
tem wounds.3,30–32 Ions have proved to be useful for wound vital-
ity and timing diagnosis, particularly Ca in early stages and Mg
in later stages.33
The aim of this study was to analyze, together and separately,
the metallic ion concentrations (Fe, Zn, Mg, and Ca) and protein
expression levels of P-selectin and cathepsin D in skin wounds in
the ligature marks to correctly characterize a vital wound in a series
of suicidal hangings where vitality was previously demonstrated.
MATERIALS AND METHODS
Human Skin Specimens
A total of 71 human vital skin injuries from ligature marks
and 71 noninjury skins of deaths by suicidal hanging were ana-
lyzed (Fig. 1). In both cases, skin specimens corresponded to skin
cross sections of 0.5 to 0.7 cm. None of the cases had experienced
severe malnutrition, malignant diseases, or metabolic disorders,
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Legaz Pérez et al
FIGURE 1. Schema of skin wounds in the ligature mark caused
by suicidal hanging, showing the location and size of the ligature
mark. The mean ligature mark size was 5  2 cm, and the specimens
were measured 0.5 to 0.7 cm.
and no substances such us cytostatic agents or glucocorticoids,
which may possibly influence wound healing, were administered
during medical treatment. In all cases, people were found dead
and skin specimens were collected at autopsy within a postmor-
tem interval between 19 and 36 hours from the discovery of the
FIGURE 2. Flowchart representing different groups of samples analyzed in this study. Numbers represent number of samples included in
each group.
2 www.amjforensicmedicine.com
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Am J Forensic Med Pathol • Volume 00, Number 00, Month 2017
body. Permission for this study was given by the relevant institu-
tional ethical committee.
Structural Damage in the Neck
Structural damage in the neck was classified from lowest to
highest level into 3 groups: (1) only the cutaneous structure af-
fected; (2) only cutaneous and subcutaneous lesions; and (3) all
main neck areas affected, including cutaneous, subcutaneous,
muscular, and bone (Fig. 2).
Determination of Metallic Ions
Human skin wounds from ligature marks (Fig. 1) and un-
damaged neck skin from the same individual were processed for
the quantification of metallic ions (Ca, Fe, Mg, and Zn) by induc-
tively coupled plasma-atomic emission spectrometry (ICP-AES;
PerkinElmer Optima 2000). A total of 0.1 g of clean dry tissue
(not formalin-fixed) was digested with nitric acid and microwave
heating (Milestone). Calibration lines of elements were made by
standard solutions (Thermo Fisher Scientific), with gradients dif-
fering for each standard solution. The detection limit of each ele-
ment in solution was measured by a blank test and the
concentrations of the metallic ions were expressed in parts per
million (ppm) of dry tissue.
Immunohistochemistry
To detect and semiquantify the protein expression of cathepsin
D and P-selectin in human skin wounds, specific immunohisto-
chemistry was used. Specimens were fixed overnight by immersion
in phosphate-buffered 4% paraformaldehyde (pH 7.4) at 4°C. Af-
ter extensive washing in 0.1 M phosphate-buffered saline (PBS;
pH 7.4), the specimens were embedded in paraffin for sectioning
into transversal skin sections using a microtome (Leica RM2255;
Germany; 60-μm thick). To analyze the cytoarchitecture, sections
were stained with hematoxylin-eosin and were also processed for
immunohistochemistry to detect cathepsin D and P-selectin. For
the immunohistochemical study, sections were deparaffinized
and incubated at 37°C for 24 hours, after blocking endogenous
peroxidase with 3% H2O2 in PBS-T (Tween 20) for 15 minutes.
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Am J Forensic Med Pathol • Volume 00, Number 00, Month 2017 Vitality in Wounds in Ligature Marks From Suicide

Sections were subsequently incubated for 30 minutes at 25°C, un-
der constant shaking with the following primary antibodies:
monoclonal mouse antihuman cathepsin D (clone DB2000,
Dako; diluted 1:400) and monoclonal mouse P-selectin (clone
1E3, Dako; diluted 1:100). After extensive washes in PBS-T, sec-
tions were incubated in a secondary biotinylated goat anti–mouse
IgG (Vector, Burlingame, Calif; diluted 1:200) for 1 hour, washed
again in PBS-T, and incubated in streptavidin biotin complex
(ABC Kit, Vector; 0.003% dilution) for 1 hour. Immunolabeling
was revealed with 0.05% diaminobenzidine (DAB; Sigma) in
0.05 M Tris buffer (pH 7.6), containing 0.03% H2O2.
Statistical Analysis
Demographic data and screening results were collected in a
database (Microsoft Access 11.0; Microsoft corporation, Seattle,
Wash), and statistical analysis was performed using SPSS 20.0
software (SPSS Inc, Chicago Ill). Pearson χ2 and 2-tailed Fisher
exact tests were ran to compare categorized variables between
groups, and a 2-sided Student t test and the nonparametric
Mann-Whitney U test were ran to compare mean values. The Pear-
son correlation coefficient was used to measure the linear associ-
ation between 2 variables. A level of P < 0.05 was accepted as
statistically significant.

Immunohistochemical Semiquantitative Analysis

Semiquantitative analysis was performed to evaluate staining
intensity by light microscopy. In each section, 5 microscopic
fields (magnification 10) were randomly selected and evaluated
for each specimen.
Cathepsin D was considered negative when no positive cells
in the section were found, moderately positive when less than 25%
of cells were positive, and strongly positive when more than 25%
of cells were positive in the ligature mark. P-selectin was consid-
ered negative when no positive cells were found in the section;
moderately positive when stained cells were observed in the epi-
dermal basal membrane and vascular walls; and strongly positive
when there was staining in the glandular cells, interstice, epidemic
basal membrane, and vascular walls.
RESULTS
Dimensional Characteristics of Ligature Mark and
Structural Damage of Neck Injuries in Deaths by
Suicidal Hangings
As shown in Table 1, the majority of deaths by suicidal hang-
ings in our study corresponded to men (86%, n = 61) with a mean
age of 35.7 ± 14.5 (years ± SD), age range of 17 to 82 years, and a
mean body weight of 75.3 ± 6.0 kg. Women represented 14%
(n = 10) with a mean age of 36.0 ± 19.3 (years ± SD), age range
of 12 to 77 years, and mean body weight of 58.2 ± 8.0 (kg ± SD).
The average dimensions of the ligature marks were as fol-
lows: depth, 0.18 ± 0.04; and width, 0.83 ± 0.40 (cm ± SD).

TABLE 1. Dimensional Characteristics of Ligature Marks and Structural Damage of Neck Injuries Depending on the Mode of
Hanging of Deaths by Suicidal Hanging

Total Males Females

N (%) n (%) n (%)
71 (100) 61 (86) 10 (14)
Mean age (mean year ± SD) 35.7 ± 15.1 35.7 ± 14.5 36.0 ± 19.3
Mean body weight (mean ± SD) 72.9 ± 8.6 75.3 ± 6.0* 58.2 ± 8.0
Ligature mark dimensions (mean cm ± SD)
Depth 0.18 ± 0.04 0.17 ± 0.05 0.20 ± 0.00
Width 0.83 ± 0.40 0.82 ± 0.37 0.89 ± 0.62
Depth/width 25.7 ± 12.13 25.01 ± 11.84 29.8 ± 13.67
Neck injuries
None 6 (8.5) 6 (9.8) 0 (0)
Subcutaneous (only) 54 (76.1) 46 (75.4) 8 (80)
Subcutaneous, muscular, and others 11 (15.5) 9 (14.8) 2 (20)
Hanging mode
Complete 42 (59.2) 37 (60.7)† 5 (50)
Neck injuries
None 3 (7.1) 3 (8.1) 0 (0)
Subcutaneous (only) 33 (78.6) 29 (78.3) 4 (80)
Subcutaneous, muscular, and others 6 (14.3) 5 (13.5) 1 (20)
Incomplete 29 (40.8) 24 (39.3) 5 (50)
Neck injuries
None 3 (10.3) 3 (12.5) 0 (0)
Subcutaneous (only) 21 (72.4) 17 (70.8) 4 (80)
Subcutaneous, muscular, and others 5 (17.2) 4 (16.6) 1 (20)
*Mean male and female body weights were compared by the 2-sided Student t test, P < 0.0001.
†Frequency of complete and incomplete hanging mode in male versus female compared by 2-sided Fisher exact test, odds ratio = 1.542; 95% confidence
interval, 0.403–5.898, P = 0.730. A level of P < 0.05 was accepted as statistically significant.

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Legaz Pérez et al Am J Forensic Med Pathol • Volume 00, Number 00, Month 2017

The depth-width ratio for the ligature marks was 25.7 ± 12.13
(mean ± SD), being similar for both sexes.
Neck injuries were related with sex and suicidal hanging
mode (complete or incomplete). Our results showed a high fre-
quency of subcutaneous neck injury (76.1%), followed by injuries
to the subcutaneous, muscular, and other structures (15.5%), and
finally the absence of neck injury (8.5%), the frequency distribu-
tion being statistically significant (χ2 < 0.001). There were no sig-
nificant differences between neck injuries according to sex or
body weight of the cadaver. The hanging mode (complete and in-
complete) was similar for both sex and body weight.
Analysis of Metallic Ion Concentration in
Ligature Mark
The concentrations of Ca, Mg, Fe, and Zn were analyzed in
71 noninjured neck skin and 71 injured skin of ligature marks
with different types of neck injuries produced by suicidal
hanging (Table 2).
Ca and Mg concentration in noninjured and injured skin
were similar (P = 0.373 and P = 0.367, respectively), but the Fe
and Zn concentrations were higher in injured skin, the differences
being statistically significant (P = 0.019 and P = 0.009, respec-
tively). The Mg/Ca, Fe/Ca, and Zn/Mg ratios were higher in in-
jured than in noninjured skin, although the differences were not
statistically significant in any case (P = 0.612, P = 0.669,
P = 0.499, respectively). Conversely, the higher Zn/Ca and Fe/
Mg ratios in injured skin represented statistically significant dif-
ferences compared with noninjured skin (P = 0.030 and
P = 0.005, respectively). In both types of neck skin, the highest
metallic ion concentrations were those of Ca (340.5 ± 248.4)
and Mg (117.9 ± 68.0), followed by Fe (66.8 ± 46.9) and Zn
(56.3 ± 68.4) (mean ppm ± SD).
When the concentrations of metallic ions were analyzed ac-
cording to the type of structural neck injury, the proportional de-
crease in Ca, Zn, and Zn/Mg levels and an increase in Fe, Mg/
Ca, Fe/Ca, Zn/Ca, and Fe/Mg levels were related with an increase
in damaged structures of the neck. By contrast, Mg concentration
was independent of types of neck injury.
Analysis of Cathepsin D and P-Selectin Expression
in Skin Wound of Ligature Marks
A total of 70 skin wound cross sections of ligature marks
were semiquantitatively evaluated with regard to the proteins ca-
thepsin D and P-selectin, and a colocalization analysis of both pro-
teins was carried out (Table 3).
Cathepsin D analysis showed that 51.3% of the analyzed
specimens presented moderately positive (38.5%) or strongly pos-
itive cells (12.8%), whereas cells were not labeled in 48.5% of the
cases. Regarding P-selectin expression, 47.2% of cells were mod-
erately (39.4%) or strongly (7.1%) positively stained in the liga-
ture marks, whereas 52.8% of the analyzed specimens did not
show positive staining. Regarding the co-expression of both pro-
teins, 19 sections (27.1%) showed a moderate degree of co-
expression and 4 sections (5.7%) were strongly positive for both
markers, whereas no co-expression was observed in 29 sections
(41.4%) (Table 3).
Finally, the expression of both proteins was analyzed in the
cross sections of skin wounds in the ligature marks, classified ac-
cording to the different neck injuries experienced (Fig. 3). No ca-
thepsin D expression was observed in 34 cases (48%) and no
P-selectin expression in 37 cases (52.8%), most corresponding to
cases where subcutaneous injury was present. Moreover, a higher
frequency of cells moderately positive to cathepsin D (Fig. 3A)
and P-selectin (Fig. 3B) was found in skin wounds with
subcutaneous injury than for subcutaneous and muscular injuries.
Analysis of Correlations Between Semiquantitative
Expression of Cathepsin D and P-Selectin and
Metallic Ion Concentration in Skin Wounds in
Ligature Marks
The expression of P-selectin and cathepsin D cells and the
concentration of metallic ions (Ca, Mg, Fe, and Zn) were analyzed
together in the skin wounds of ligature marks (Fig. 4).
Statistically significant correlations were found between ca-
thepsin D and Fe (R = 0.440, P < 0.001; Fig. 4A). Low Fe levels
were observed in the case of a negative expression for cathepsin D,
whereas high levels of Fe were found in sections with strong

TABLE 2. Analysis of Metallic Ion Concentrations in Noninjured and Injured Wound Skin of Ligature Mark Associated With Different
Types of Neck Injury in Deaths by Suicidal Hanging

Types of Neck Injuries

Metallic Noninjured Injured Skin Cutaneous Cutaneous and Cutaneous, Subcutaneous,
Ions* Skin of LM Only Subcutaneous and Muscular
(Mean ± SD) N = 71 N = 71 P† n=6 n = 54 n = 11
Ca 372.1 ± 167.7 340.5 ± 248.4 0.373 429.1 ± 41.4 222.1 ± 103.3 122.8 ± 32.1
Mg 131.1 ± 110.1 117.9 ± 68.0 0.367 102.6 ± 69.4 95.69 ± 67.8 113.3 ± 56.3
Fe 50.8 ± 32.1 66.8 ± 46.9 0.019 47.2 ± 23.6 62.2 ± 47.5 72.5 ± 39.4
Zn 32.7 ± 29.1 56.3 ± 68.4 0.009 96.7 ± 77.4 48.5 ± 71.2 56.1 ± 47.2
Mg/Ca 0.38 ± 0.65 0.45 ± 0.95 0.612 0.30 ± 1.83 0.49 ± 0.53 0.51 ± 0.49
Fe/Ca 0.28 ± 0.22 0.32 ± 0.75 0.669 0.11 ± 0.57 0.28 ± 0.46 0.59 ± 1.23
Zn/Ca 0.12 ± 0.17 0.37 ± 0.94 0.030 0.24 ± 0.24 0.34 ± 1.27 0.53 ± 1.31
Fe/Mg 0.43 ± 0.34 0.66 ± 0.58 0.005 0.46 ± 0.34 0.65 ± 0.70 0.64 ± 0.70
Zn/Mg 0.36 ± 0.26 0.41 ± 0.56 0.499 0.88 ± 0.56 0.43 ± 0.57 0.43 ± 0.57
*Metallic ion concentration was quantified in parts per million. Means metallic ion concentrations were compared by the 2-sided Student t test.
†P value used to compare noninjured and injured skin of ligature mark. A level of P < 0.05 was accepted as statistically significant and are shown in bold.
LM, ligature mark.

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Am J Forensic Med Pathol • Volume 00, Number 00, Month 2017
TABLE 3. Analysis of Cathepsin D and P-Selectin Expression in Injured Skin of Ligature Mark of Death by Suicidal Hanging
Negative
34 (48.5)
Negative 29 (41.4)
37 (52.8)
P-Selectin, n (%) Moderately Positive 5 (7.1)
N = 70 28 (39.4)
Strongly Positive 0 (0)
5 (7.1)
cathepsin D immunoreactivity (P < 0.0001). Similar behavior was
observed for P-selectin and Fe (R = 0.319, P = 0.007; Fig. 4E). By
contrast, no significant relationships were found between Ca, Mg,
and Zn and the expression of cathepsin D (Fig. 4, B–D) or
P-selectin (Fig. 4, F–H).
DISCUSSION
In this study, a large cohort of human vital wounds of the lig-
ature mark of cadavers from suicidal hangings was studied, ana-
lyzing, together or separately, the metallic ions concentration of
Fe, Zn, Mg, and Ca and the expression of P-selectin and cathepsin
FIGURE 3. Semiquantitative analysis of cathepsin D and P-selectin
in skin wounds in the ligature mark with different internal neck
injuries. A, Number of cases with cathepsin D positive cells in the
ligature mark associated with neck injuries. B, Number of cases
with P-selectin positive cells in the ligature mark associated with
neck injuries. White bars designate no injury; gray bars indicate
cutaneous and subcutaneous injuries; and solid bars represent
subcutaneous and muscular injuries. Statistical analyses
were performed using Pearson χ2. P < 0.05 value was
considered significant.
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Vitality in Wounds in Ligature Marks From Suicide
Cathepsin D, n (%)
N = 70
Moderately Positive Strongly Positive
27 (38.5) 9 (12.8)
7 (10.0) 1 (1.4)
19 (27.1) 4 (5.7)
1 (1.4) 4 (5.7)
D proteins. This is the first time that both types of biomarker have
been studied together to characterize such a large cohort of human
vital wounds of this type.
The average age of the subjects of both sexes in the study was
35.7 years, which is similar to that mentioned in other studies.34,35
Similarly to other studies, too, there were more male suicides than
female.35–37 Analysis of the ligature mark dimensions and struc-
tural neck injuries by hanging shows that, despite weight differ-
ences between the sexes, neither parameter was affected by the
mode of hanging (complete or incomplete).
Most of the neck injuries that occurred affected only subcu-
taneous structures, not muscle or bone structures, whereas other
studies observed that neck structure fracture was predominant in
complete hanging and in older subjects.38–43 Other studies pre-
sented contradictory results regarding sex, suspension type, or lig-
ature type.39,41 These variations in the literature could be the result
of a lack of a common method for examining neck structures and/
or variability in assigning grades to any damage, thus affecting the
interpretation of the results.
On the other hand, several studies have described that inor-
ganic elements play a critical role in several aspects of angiogen-
esis, for example, in healing and regeneration processes.44–46
Our results show that calcium is the most abundant metallic ion
in the injured skin of the ligature marks studied, followed by
Mg, Fe, and finally Zn, the high level of calcium perhaps being
due to it being a component of the epidermal response to
wounding, regulating stem cell activation through increased
β-catenin transcriptional activity.45 Nevertheless, calcium levels
decreased as wound injuries in the cadaver increased, perhaps
due to the interruption of the process of healing because the dying
process was faster in these cases.
Our findings point to an increase in Fe and a decrease in Zn
concentrations injured skin with subcutaneous and muscle layer
affected compared with cutaneous layer affected, corroborating
the observations of other studies, which also found increased Fe
concentrations in antemortem wounded skin and muscle but no
difference in the metal ions Zn and Mg.30 Besides, studies realized
in pig vital wounds also found higher concentrations of Fe in an-
temortem wounded skin than in postmortem wounds influenced
by putrefaction.32 On the other hand, our data show that concen-
trations of Zn decrease as the severity of neck damage increases,
perhaps due to the ability of this metal to reduce the expression
of proangiogenic growth factors while promoting the production
of antiangiogenic factors such as endostatin.44
A high percentage of the samples analyzed showed a nega-
tive expression of cathepsin D, confirming the results obtained
by other authors in fragments of human abdominal skin.47 More-
over, the high expression of cathepsin D found in our series
would be due to the postmortem period elapsing until the body
was found and the sample obtained. High values of cathepsin D
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Legaz Pérez et al Am J Forensic Med Pathol • Volume 00, Number 00, Month 2017

FIGURE 4. Correlation between metal ion concentrations and cathepsin D and P-selectin expression in the ligature mark. Correlations
between levels of Fe (A), Ca (B), Mg (C), and Zn (D) and semiquantitative expression of cathepsin D. Correlations between levels of Fe (E), Ca
(F), Mg (G), and Zn (H) and semiquantitative expression of P-selectin. P < 0.05 values were considered significant and are indicated in the
graphs. R indicates Pearson correlation coefficient. All values considered were represented as mean concentration expressed in part per
million (ppm) in dry skin.

were also observed by Montisci et al27,47 in postmortem human
skin fragments.
Similarly, a high percentage of specimens with a negative ex-
pression of P-selectin was found in our series of vital wounds per-
haps as a result of postmortem injuries to endothelial cells48 or a
decrease in P-selectin staining intensity due to the postmortem pe-
riod because P-selectin staining is known to increase up to a wound
age of 1 hour, before decreasing.23 On the other hand, the positive
expression found may be a result of damage that occurred during
life, corroborating observations made by Wyss et al,48 or to the high
frequency of moderate subcutaneous skin injuries, because moder-
ate intensity of P-selectin in noninjured skin has been observed in
previously studies.23 Perhaps these results arise because it is diffi-
cult to control the postmortem time in suicidal hangings.

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Am J Forensic Med Pathol • Volume 00, Number 00, Month 2017
Thus, our data identify high concentrations of Ca and Mg
compared with Fe and Zn in human vital wounds in the ligature
marks of suicidal hanging. Fe and Zn concentrations are signifi-
cantly higher in injured skin than noninjured skin. Ca and Zn de-
creased and Fe concentrations increased in the ligature marks with
the severity of neck injury. In injured skin, a high percentage of
negative and moderate expression of both cathepsin D and
P-selectin were observed and correlated with a low or medium
concentration of Fe.
In conclusion, the joint study of metallic ions and proteins al-
low to characterize and to differentiate an injured vital wound of
noninjured skin, especially when the damage in the tissue affects
to the majority of the structures of the skin, but despite of the dif-
ficulty involved in obtaining human samples of this type, it would
be interesting to contrast our findings based on studies of protein
expression quantification and other biomarkers in human samples
experiencing neck wounds, while carefully trying to control the
postmortem interval, to safely differentiate between vital and
postmortem wounds.
ACKNOWLEDGMENTS
The authors are grateful to the Institute for Legal Medicine,
Sao Paulo, Brazil, and the Institute for Legal Medicine, Coimbra,
Portugal, for their rewarding collaboration in this research study.
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