Advances in the Diagnosis of Wound Vitality: A Review

Author(s):
Hernndez-Cueto, Claudio M.D., Ph.D.; Girela, Eloy M.D., Ph.D.;
Sweet, David J . D.M.D., Ph.D.
Issue: Volume 21(1), March 2000, pp 21-31
Publication Type: [Articles]
Publisher: 2000 Lippincott Williams & Wilkins, Inc.
Institution(s):
From the Department of Legal Medicine, Faculty of Medicine,
University of Granada, Granada, Spain (C.H.-C., E.G); and the Bureau
of Legal Dentistry, University of British Columbia, Vancouver, Canada
(D.J .S.).
Manuscript received March 24, 1999; accepted J uly 14, 1999.
Address correspondence and reprint requests to Claudio Hernndez-
Cueto, Departamento de Medicina Legal, Facultad de Medicina,
Universidad de Granada, 18071 Granada, Espaa; email:
chc@goliat.ugr.es .
Keywords: Wounds, Injuries, Vitality diagnosis, Macroscopic diagnosis, Biochemical diagnosis,
Enzyme-histochemical diagnosis
Table of Contents:

Abstract
The diagnosis of the vital origin of wounds in many cases remains an unsolved problem for the
forensic pathologist. Practical experience enables the expert to diagnose the vital or postmortem
origin of wounds on the basis of macroscopic examination. In some cases, optic microscopy is
used to confirm the diagnosis. In many other cases, additional more sensitive and specific
markers of vitality are required. In the past 50 years, comprehensive research on this topic has
resulted in a better understanding of the acute inflammatory reaction. The development and
application of sensitive and specific markers through research in the areas of histochemistry,
enzymology, and biochemistry has provided a partial solution to the problems involved in
wound vitality diagnosis. A review of this challenging area of forensic pathology, including an
explanation of these methods and markers, is presented in this paper.


The diagnosis of the vital origin of wounds in many cases remains an unsolved problem for the
forensic pathologist. Nevertheless, in the past 50 years, the development of histochemistry,
enzymology, and biochemistry, and the application of these techniques to the diagnosis of
wound vitality, has allowed a partial solution to this problem.
Medical knowledge regarding the vitality of wounds was previously confined to macroscopic
findings reported by the physician A. C. Celso (1) in the first century and other complementary
studies such as optic microscopy. Celso described the "acute inflammatory reaction" as the
manner in which living tissues react to injuries. The period between this description and optic
microscopy findings was 19 centuries. These findings, as well as the recent development of
several markers, are based on a common feature of living tissues: the existence of a "vital
reaction" to any insults.
VITAL REACTION (ACUTE INFLAMMATORY REACTION)
One characteristic of living organisms is the ability to respond in the presence of an external
stimulus. When this stimulus is a traumatic offense, whether biologic, physical, or chemical, the
tissue response consists mainly of an inflammatory reaction that is proportional to the
magnitude of the tissue offense. The inflammatory reaction consists of a complex mechanism,
the objective of which is destruction of the noxious stimulus and tissue repair.
This phenomenon was described by Hunter in 1793: "Inflammation, of any cause, is an effect
destined to restore the natural function of affected parts, and it should not be considered as an
illness, but a beneficial process which appears after the noxious stimulus goes down" (1,2). In
fact, the inflammatory reaction is a complex phenomenon that is not well understood. It consists
of several apparently different processes such as enzymatic and biochemical induction,
hemodynamic and vascular changes, and cellular activity, among others. All of these processes
are aimed at destruction of the noxious stimulus and effecting tissue repair. The vital reaction
also includes other related events which are not strictly considered part of the inflammatory
response, such as platelet aggregation, complement activation by coagulation factors, and
metabolism of prostaglandin. The vital reaction, as described by Strassman in 1954 (1,2) and
according to a previous definition by Plenk in 1786, is "... one of organs and tissues, which
needs for development the presence of living cells" (Fig. 1). This kind of reaction does not
happen in nonliving tissue (Fig. 2).



FIG. 1. Cellular infiltrate shows vital origin by rutin light
microscopy.




FIG. 2. Postmortem incised wound inflicted 15 to 30 minutes after death. Vital
reaction is not clear; the doubtful diagnosis may be resolved by biochemical
markers (i.e., histamine, serotonin, and cathepsin D).

In forensic case work there are many situations dealing with skin injuries in which the
differentiation between vital or nonvital origin is of paramount importance in the establishment
of the cause and manner of death. Practical experience enables the forensic pathologist to make
the diagnosis of the vital or postmortem origin of wounds on the basis of macroscopic
examination. If the wound was obviously inflicted on a living individual, the injured tissues,
mainly skin, will show the typical inflammatory response as summarized by Legrand du Saulle
in the past century (3) (Table 1). Conversely, if the wound was inflicted on a deceased
individual, these macroscopic features will be absent due to the absence of a vital reaction. If the
wound was inflicted close to the moment of death, it can be difficult to make such a diagnosis.
It is well established that there is no exact boundary between life and death. The period between
life and cellular death is variable and depends on factors such as the cause of death, individual
susceptibility, and duration of pain, among others. Furthermore, different tissues die at different
rates depending on their ability to support anoxia. If, during the period between life and cellular
death, the body suffers any insult, a vital-like reaction may be seen, although usually it is of
reduced intensity. This reaction has been called an "agony reaction" or "intermediate reaction."
It is indicative of the lack of precision of the tissue reaction and results in uncertainty as to the
diagnosis of wound vitality. For this reason, it is difficult to differentiate between the vital or
postmortem origin of injuries sustained close to the moment of death. Tourdes (1-3) described
an "uncertainty period" of approximately 6 hours around the time of death in which it is not
possible to establish the vital or postmortem origin of a wound.


TABLE 1. Scheme of Legrand du Saulle for the diagnosis of
vital and postmortem wounds

The goal of many forensic researchers has been to shorten this period. Many new techniques
have been applied during the last century. After using macroscopic methods exclusively, optic
microscopy was applied to allow recognition of early vital reaction changes, especially those
derived from a cellular response during the acute inflammatory reaction. The work of
Piedelievre is especially relevant (1-3). He was the first to take into account the leukocyte
infiltration of the damaged site as a sign of vital reaction (Fig. 1). However, in 1921, the degree
of intensity of leukocyte flow to be considered as a vital response was controversial (2,4).
Similarly, changes in red blood cells and hemoglobin have been studied (3), although with poor
results. Additionally, changes in the capillary network were studied by Lo Menzo et al. (5,6),
and features of the vital clot network were studied by Bhm using scanning electron microscopy
(2,4).
These studies have significantly contributed to reduction of the "uncertainty period" described
by Tourdes. Development of immunochemistry, histochemistry, enzymology, and biochemistry
techniques and the application of these methods since the 1960s are responsible for the present
status of diagnostic ability. The uncertainty period has been reduced to minutes.
ENZYME-HISTOCHEMICAL MARKERS
Enzyme Activities
Main developments in this aspect of vitality diagnosis were undoubtedly due to studies by
Raekallio, a professor of forensic medicine at the University of Turku, Finland. He not only
pioneered the application of enzyme-histochemical techniques but also developed many of the
essential markers used for the diagnosis of wound vitality. During the 1960s, he reported
changes in enzymatic activity at the edges of vital wounds. These changes can be recognized
even a short time following wound infliction.
Vieira noted that functional disturbances occur before morphologic changes in the inflammatory
reaction (7). These functional disturbances include the release of lysosomes or other cell
constituents involved in the acute inflammatory reaction. This results in an increase in
enzymatic activity at the damaged site. Clark showed that necrotic or degenerative tissues
produce the opposite phenomenon (8).
Histochemical demonstration of an enzyme is based on its catalytic activity on a specific
substrate. Under appropriate circumstances, the products of the reaction form an insoluble
deposit which can be seen on its own or through the use of stains.
Using enzyme-histochemistry methods, Raekallio studied the behavior of several enzymes,
including alkaline phosphatase, acid phosphatase, arylaminopeptidase, esterase, and adenosine
triphosphatase (9-23). He noted changes in enzymatic activity in vital skin wounds which did
not happen in nonvital wounds. These conclusions resulted in the identification of two clearly
delineated zones of differing enzymatic activity around a vital wound: a central zone and a
peripheral zone.
The central zone is an area 200 to 500 wide that is located at the edges of a vital wound and
shows a gradual decrease in enzymatic activity. This decrease can be detected between 1 and 4
hours after wound infliction. This zone reflects the imminent necrosis that will occur, although
this necrosis does not become histologically evident for up to 32 hours after wound infliction.
This enzymatic response has been called the "negative vital reaction." It is recognized as the
maximum traumatic offense and tissue destruction zone. As a consequence of capillary damage,
there is a extreme lack of oxygen, which results in a quick and severe reduction in local pH.
These changes give rise to proteolytic enzyme activation which is destined to complete the
destruction of damaged tissues as a first step in the acute inflammatory reaction.
The peripheral zone is an area 100 to 200 wide circumscribing the central zone. This area
shows a remarkable increase in enzymatic activity 1 hour after wound infliction. Histologically,
it is a focus of leukocytic flow, particularly of polymorphonuclear leukocytes, which are
subsequently replaced by mononuclear leukocytes, lymphocytes, and active fibroblasts. These
components constitute an active repair step of the acute inflammatory reaction. This response
has been called the "positive vital reaction."
The second important finding of Raekallio was the realization that the increase in enzymatic
activity in the peripheral zone occurs over a specific time interval, and this time interval is
different for each enzyme. Furthermore, these enzymatic activities possess good postmortem
stability and can be seen up to 5 days after death (24). For these reasons, enzymatic activity is
useful in determining the age of a wound.
Both the central and the peripheral zones, which are histologically demonstrable in old injuries
(8-16 hours postinfliction), are not present in post-mortem wounds, at least those inflicted 1
hour or more after death. Esterases and adenosine triphosphatase are the first enzymes to
increase, at about 1 hour after wound infliction. Aminopeptidase activity increases 2 hours after
infliction, whereas alkaline phosphatase does not increase until 4 hours after infliction. Acid
phosphatase shows a late increase, approximately 8 hours after wound infliction. This sequence
of increasing enzymatic activity gave rise to a "biological timetable" reported by Raekallio (25-
28), and we corroborated his results in human autopsy samples (25). Subsequently, similar
results were obtained by others (29-48).
The most significant of these reports is that of Fatteh (35), who studied the following enzymes
in an experimental series: esterases, leucinaminopeptidase, acid phosphatase, alkaline
phosphatase, DNA polymerize, and RNA polymerize. Similar studies on these human skin
constituent markers were carried out by Pullar in 1973 (49) and by others (22,50-52). These
studies report the sequence of increasing enzymatic activities as follows:
* esterases, 10 minutes
* acid phosphatase, 1 hour
* alkaline phosphatase, 3 hours
* leucinaminopeptidase, 3 hours
* DNA polymerizes at 4 hours
* RNA polymerizes at 4 hour
* Different zones of enzymatic activities also were distinguished:
* edges of wound, 25-50 in depth
* external zone, 100-400 in depth
* internal zone, 100-400 in depth
Therefore, the following conclusions can be inferred from these studies:
1. Enzymatic reactions in wounds are specific indicators of wound vitality.
2. Studies involving different anatomic regions yield similar results.
3. In autopsy samples, results are reliable as late as 5 days after death.
There are many factors that can influence enzymatic activity. Raekallio (17) and Russell (53)
studied the influence of age on histochemical enzyme detection in relation to vitality diagnosis
using experimental animals. It was determined that there were no changes in enzyme behavior
in relation to age except in the "peripheral zone," where the increase in enzymatic activity is
smaller for older animals. This finding was also noted in human subjects and corroborated by
Berg (31). Other factors that likely influence the enzymatic response are poor relative health and
the presence of multiple and severe injuries.
Similar research was conducted by several other authors. Pioch studied esterase activity in
contusions (45,46), Malik studied esterase and aminopeptidase in animal wounds (43), Tanaka
studied esterase in human wounds (47), and Zhu and Wang (48) studied esterase in
experimental wounds.
Other Histochemical Studies
Similar results were obtained by Lo Menzo and Marziano (5) using fluorescence microscopy.
When stained with acridine orange, vital wounds of at least 1 hour duration showed a greenish
yellow fluorescence at the edges and base of the wound. Maximum fluorescence was reached in
wounds of 8 hours' duration. This is thought to occur as a result of nucleic acid release
following tissue necrosis.
The behavior of nucleic acids has been studied by several authors. Bunting and White reported
in 1950 that fibroblast regeneration causes an increase in RNA (54), whereas nuclear DNA did
not vary remarkably (55). The greatest RNA concentration appears at 7 to 9 days after wound
infliction.
Buris and Kiss (56) and Pullar (49) noted a significant increase in cytoplasmic RNA in vital
wounds after 3 hours. This is in keeping with the results reported in 1961 by Raekallio (25), in
which cytoplasmic RNA increased in the peripheral zone of vital wounds of 32 hours' duration.
Additionally, both RNA and DNA decreased in connective tissue cells 16 hours after wound
infliction, and in epidermis cells after 64 hours. These vitality-related changes are evident as late
as 5 days after death, and they do not occur in nonvital wounds.
Finally, in studies involving experimental animals performed between 1988 and 1991,
Oehmichen et al. (57-61) observed an increase in RNA synthesis in basal cells at the edges of
vital wounds of 10 hours' duration. Additionally, there was an increase in DNA among these
cells in vital wounds of 20 hours' duration and an increase in nucleic acids in cadaveric tissues.
This report resulted in controversy regarding the usefulness of this measurement.
Using histochemical methods, the behavior of glycosaminoglycans has been studied in relation
to vitality diagnosis. Dunphy and Udupa (62), Raekallio (14), Lindner (39), and Nevel and Gee
(63) reported that glycosaminoglycans undergo a decrease in the central zone of vital wounds up
to 32 hours after infliction, whereas they increase in the peripheral zone at a depth of up to 100
to 300 . This phenomenon has also been noted in cases of mechanical asphyxia, particularly by
strangulation and hanging, as well as contusions.
Since the studies of Orsos in 1935 (64) on connective tissue disturbances in wounds, further
investigations of metachromic modifications have been completed. These modifications have
been observed in the peripheral zone of vital wounds by Krauland in 1955 (65) and Lindner in
1982 (41).
Other nonenzymatic markers of wound vitality have also been studied. Durigon et al. (66) and
Maeno et al. (67) noticed increases in C3 factor, fibrin, and immunoglobulins G, A, and M in
wounds older than 10 minutes. Laiho (68,69), and later Geiler (70), studied the behavior of
fibrin. They realized that fibrin formation occurs in wounds produced 7 hours after death.
Eisenmenger et al. (71) studied immunohistochemical changes in collagen in relation to the
vitality diagnosis of wounds. Protease inhibitors such as [alpha]
1
-antitrypsin, C1, antithrombin
III, alfa-1-antichymotrypsin, [alpha]
2
-antiplasmin, [alpha]
2
-macroglobulin, and protein C have
been used successfully as early markers of wound vitality in both animal and human subjects by
Oehmichen et al. (58-60,72,73).
Wound vitality markers have also been studied in tissues other than incised skin injuries.
Karkola (74) noted a decrease in enzymatic activity of the damage focus in liver wounds 8 to 10
hours after infliction. In 1973, Todo (2) studied tissue reactions after dental extractions. He
noticed a moderate increase of enzymatic activity on the fifth day after extraction, especially for
acid phosphatase, alkaline phosphatase, and oxidoreductase. There was a second, higher
increase between the tenth and twelfth day after extraction at the same time that fibroblasts were
transformed to osteoblasts. These injuries were studied by Plagman (2), although he focused on
other enzymes such as succinate dehydrogenase, lactate dehydrogenase, and acid phosphatase.
Esterase activity has been studied in different injuries and tissues. Pioch (46) and Berg and Edel
(32) studied esterase activity in experimental contusions; Buris et al. (75) and Karlsmark et al.
(76) performed similar studies in electrical lesions; Ross and Walker (77), Somogyi et al. (78),
Malik (43), and Nanney (79) studied esterase activity in burns; Fechner et al. (80) performed
studies in muscular injuries; Kampmann et al. (81,82) performed studies in incised liver
wounds; and Raekallio et al. (52,83-85) studied activity in bone fractures.
Most authors accept these findings. However, there are some who are skeptical of the usefulness
of enzyme-histochemical markers to determine the timing of wounds and the differentiation
between vital and postmortem wounds. Dotzauer and Tomaska (86) and Hou-J ensen (87) have
published in relation to this skepticism (88).
Despite the limitations of these markers, it is fair to say that they have contributed to reducing
the "uncertainty period" substantially, at least in wounds older than 1 hour, using easy, quick
and inexpensive techniques. Recently, much effort has been expended on the use of
histochemistry (mainly immunohistochemical methods) in studying factors involved in cellular
regeneration and wound vitality. Between 1992 and 1995, Betz et al. studied the synthesis of
type IV collagen (89-98). Much of this work was based on the previous experience of
Eisenmenger et al. from the Institute of Legal Medicine of Mnchen (71). Eisenmenger studied
heparin sulfate in myofibroblasts, [alpha]
1
-antichymotrypsin, fibronectin, and cytokeratins in
incised wounds in human skin. Betz et al. (97), using immunohistochemical methods, studied
several markers of macrophage maturation which are indicative of different steps of
inflammation. These included early markers such as 27 E 10, intermediate markers such as RM
3/1, late markers such as 25F9, and chronic markers such as G 16/1. Initial results are promising
because of the potential usefulness of these markers in determining the age of wounds, although
it may be too early for definitive conclusions.
Fechner et al. (80), from the University of Mnster, studied actin and myoglobin levels in
muscular injuries in animal and human samples using polyclonal antibodies. They noted a
decrease in these proteins in vital muscular injuries which did not occur in postmortem injuries.
Myoglobin was the first to decrease, and it is detectable up to 72 hours after death.
Continuing the studies on cellular proliferation, Oehmichen and Crpelin (88) reported a new
wound vitality marker called bromodeoxyuridine. This compound is incorporated by the cell
during DNA synthesis and allows recognition of vital wounds up to 32 days after death.
Althoff (4) disagrees with the usefulness attributed to these markers. He noticed that collagen
undergoes postmortem changes, termed fibrinoid-like degeneration, as a result of chemical or
environmental influences. His opinion is that more rigorous investigations are needed before
these markers can be applied to the vitality of wounds.
BIOCHEMICAL DIAGNOSIS OF THE VITALITY OF WOUNDS
The use of biochemical markers for the past 35 years has allowed a substantial reduction in the
"uncertainty period" described by Tourdes. In addition to the work of Tarsitano (4,25) which
dealt with the increase in chloride levels in intravital damaged tissue which requires at least 5
hours to develop, many other biochemical markers whose changes may be detected during the
immediate perimortem interval have been studied.
The work of Raekallio is the most significant with regard to biochemical markers. He concluded
that the reaction of damaged tissues to noxious stimulation is immediate; therefore, the problem
is to find early biochemical markers that are sensitive enough to reveal the initial stages of the
inflammatory reaction.
Vasoactive Amines
Histamine and serotonin are both vasoactive compounds known to participate in the initial
stages of the acute inflammatory reaction. From numerous studies it has been deduced that
histamine is responsible for the initiation of vascular changes seen in inflammation. Conversely,
serotonin has been demonstrated in early inflammatory exudates. In many cases, both
compounds are released simultaneously, as happens in the initial stages of a traumatic injury.
In 1965, Fazekas and Viragos-Kis (99) noticed an increase in free histamine in the ligature
marks found after vital hanging. These results were later corroborated by Berg et al. (30). From
these studies it can be concluded that histamine levels show a 50% increase in vital skin wounds
that are inflicted at least 20 to 30 minutes before death, whereas serotonin levels increase up to
100% in wounds inflicted less than 10 minutes before death.
Raekallio and Mkinen (100), Berg and Bonte (33), Fatteh (101,102), Karkola and Raekallio
(103), and Sivaloganathan (104) correlated changes in histamine and serotonin with different
stages of wound aging:
* 5 minutes: slight increase or decrease of histamine, increase of serotonin
* 5-15 minutes: increase in histamine greater than increase of serotonin
* 15-60 minutes: increase in serotonin greater than increase of histamine
Many authors have studied the factors that influence these changes in histamine and serotonin
levels. Kampmann et al. (82) described the influence of acids and bases on the initial stages of a
skin reaction after mechanical injuries and reported that the cellular reaction and enzymatic
responses were different. Leukocyte infiltration into damaged tissues after mechanical trauma is
less affected by the influence of these chemical compounds (particularly bases) than enzymatic
activities at the edges of a wound.
In undamaged skin there is an increase of histamine in the presence of acidic conditions,
whereas serotonin clearly decreases. Conversely, acidic conditions in incised skin cause a drop
in histamine levels and a significant rise in serotonin levels.
It is also worth mentioning the recent work of Maeno et al. (105,106) in which high-
performance liquid chromatography (HPLC) was used for measurements of histamine and
serotonin levels in wounds, as well as norepinephrine (NE), 3,4-dihydroxyphenyl acetic acid
(DOPAC) and 5-hydroxyindol acetic acid (5-HIIAA). The usefulness of these new markers is
still controversial.
Recently, a paper was published by researchers from the University of Santiago de Compostela
in which spectrophotometric and spectrofluorometric methods for the determination of
histamine and serotonin in skin samples of rabbits and humans were described (107). The
spectrophotometric method was found to be superior in terms of reproducibility and sample
stability.
Catecholamines
Eranks (4,25) used fluorescence methods to demonstrate the presence of norepinephrine.
Subsequently, catecholamines were used in forensic pathology in wound aging applications.
Penttila (108) proved that by using this method he could identify wounds produced up to 60
minutes before death. Unfortunately, this fluorescence disappears 8 to 16 hours after death.
Enzymes
Enzymatic markers of wound vitality have been extensively studied. J arecki (109) found
differences in enzymatic activity between vital wounds of a minimum age of 30 minutes and
postmortem wounds by using isoelectric focusing of esterases in polyacrylamide gels. Raekallio
(16) corroborated these results in relation to aminopeptidases. Laiho (69) discovered increases
in peroxidase activity in vital skin wounds produced 30 minutes before death.
Brown et al. (110,111) studied the behavior of lysosomal enzymes in vital contusions and
reported increased activity 2 days after trauma which reached a maximum level between 3 and 5
days after trauma.
Cathepsin D (E.C. 3.4.23.5) has been studied in both experimental and human wounds (4,112-
118). Cathepsin D is the main acid proteinase in most animal tissues. It is activated at the focus
of damage to digest dead cells in the low pH induced by hypoxia and necrosis.
Spectrophotometric measurements of cathepsin D allow the diagnosis of the intravital origin of
wounds within as little as 5 minutes. Other studies on cathepsin A and B have also been
completed, although the results are not conclusive. We, in collaboration with the University of
Heidelberg, have evaluated the use of cathepsin D levels to determine time of wounding in
humans. The behavior of cathepsin D has been compared with that of vasoactive amines.
Studies have been completed on the postmortem stability of cathepsin D up to 48 hours after
death.
Ions
We studied other wound vitality markers using atomic absorption spectrophotometry in both
experimental and human wounds up to 48 hours after death, including various ions (Ca, Mg, Cu,
Zn, Fe, Na, and K) (4,119-121). We compared the efficacy of ion markers with that of
vasoactive amines. Ions have proved to be useful for the diagnosis of the vitality and timing of
wounds, particularly Ca in the early stages and Mg in the later stages (>6 hours). Similar results
have been reported by others (122-124).
Prostaglandins
Prostaglandins are released by traumatized cells as mediators of functional hyperemia and
inflammatory response. There is evidence of their participation in the wound-healing process,
particularly PGF2b and PGE2.
Prostaglandins have been studied in burns (20) and wounds (125). In 1988, Lasarov et al. (126)
noticed that there was an increase in PGF2b levels in vital wounds produced 10 to 60 minutes
before death. These amounts were up to 100% greater than those found in postmortem incisions.
Contrary to these investigators, after studying a series of human skin samples, we concluded
that PGF2b is not a useful biochemical marker for the differential diagnosis of vital versus
postmortem cutaneous wounds. These results are a consequence of a collaboration with Vieira
et al. from the University of Coimbra (127). Studies of PGF2b and PGE2 were completed using
radioimmunoassay and radioimmunoassay plus mass spectroscopy-gas chromatography. As was
previously observed for PGF2b, the results with PGE2 show great variability. The postmortem
interval has a significant influence on these levels, as does prostaglandin regulation mechanisms
which are not well understood. In our opinion, this raises serious doubt regarding the reliable
use of prostaglandins as markers of wound vitality (127).
Coagulation Compounds
Studies involving the fibrin network, clot formation, and subsequent clot breakdown have been
used to determine the age of vital and postmortem wounds. The work of Bhm (4) using optic
and electronic microscopy is significant in this regard.
The tissue repair process may be determined by biochemical methods. We recently completed a
study to show this using D-dimer (DD), an intermediate product of fibrin metabolism (128). DD
is an epitope that is present only after stabilization, or cross-linking, of the fibrin network and
subsequent lysis by plasmin. We studied DD using an enzyme-linked immunosorbent assay
(ELISA) from Asserachrom (Paris, France) in incised wounds (129), abrasions, and contusions.
It was determined that DD is useful in the determination of the vitality of incised wounds
because it increases up to 100% in vital wounds produced at least 5 minutes before death in
comparison with control samples.
CONCLUSIONS
Currently, forensic pathologists are able to establish the differential diagnosis between a vital
skin wound and a postmortem one by the use of a wide battery of markers. These markers have
significantly contributed to reducing the "uncertainty period" described by Tourdes to nearly
zero. Nevertheless, most cases are solved by the expert on the basis of macroscopic
examination, with confirmation by optic microscopy when the postmortem interval makes this
possible. In some cases, additional studies are necessary to obtain a more exact diagnosis of
vitality, such as "compulsory" and usually definitive measurements of histamine, serotonin, and
cathepsin D levels in the edges of the wound to compare them with levels of these markers in
homolateral control zones.
Although all these markers are currently still under investigation, there are many previous
experiences on human specimens that show the usefulness of these markers in the diagnosis of
vitality of skin wounds. Although in some cases their use in a judicial report may be disputed, it
is clear that these tests can contribute essential information for case resolution. The markers
described in this article are reasonably quick, inexpensive, and available for most forensic
laboratories. Because of the fact that most of the research on vitality diagnosis markers was
performed using incised skin wounds, it will be of value to enlarge and apply these results to
other traumatic lesions such as bone fractures.
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