Forensic Science International

113 (2000) 153–164 www.elsevier.com / locate / forsciint

Forensic wound examination

T. Ohshima*
Department of Legal Medicine, Kanazawa University, Faculty of Medicine, Takara-machi 13 -1, Kanazawa 920 -8640, Japan

Abstract

Wound examination is of prime importance in forensic pathology, and it is desirable to establish a wound examination
system in order to evaluate and record the nature of wound more accurately and objectively. Modern diagnostic techniques
and devices as well as advanced cell-biological methods should be introduced as the means for this aim. For example,
radiological, endoscopic or magnetic resonance imaging (MRI) examination have been used in addition to examination with
the naked eye. In our department, a binocular surgical operating microscope is routinely employed at forensic autopsy, which
is useful for elucidating the nature of wound in more detail. It is also necessary to determine whether a wound has vitality,
and, if antemortem, how long before death the wound has been sustained. For the determination of wound age including
vitality, various biological factors such as cytokines and extracellular matrix components involved in wound healing have
been examined by histopathological methods. Our studies have shown that interleukin (IL)-1a, IL-1 b, IL-6, IL-10 and tumor
necrosis factor-a are possibly useful markers for wound age determination as well as cell-biological indicators of vitality.
Furthermore, molecular biological techniques have been intended to be applied to wound examination; our experimental
study has shown that even mRNA of cytokines mentioned above can be histologically detected by reverse transcriptase–
polymerase chain reaction or in situ hybridization. A trial of forensic wound examination from macroscopic to molecular
level is discussed.  2000 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Forensic pathology; Wound examination; Cell biological methods

1. Introduction including basic studies on wound examination,

should be directed to forensic practices. In other
In forensic pathology, it is needless to say that words, studies without forensic aspects are not so
wound examination is the most important matter and valuable and might be even complacent.
that it requires much experience of forensic practice.
It is indispensable to establish the comprehensive
and systemic wound examination procedure. This 2. General aspects of wound examination
means, in order to exactly diagnose the cause and
manner of death, it is essential to describe the 2.1. Definition and type of wound from the
findings of wounds correctly and objectively as much viewpoints of forensic pathology
as possible. In fact, forensic studies themselves,
Knight [1] defines ‘wound’ in his famous text-
*Tel.: 181-76-265-2220; fax: 181-76-234-4234. book. Namely, a wound or injury can best be defined
E-mail address: ohshimat@med.kanazawa-u.ac.jp (T. Oh- as ‘damage to any part of the body caused by the
shima). application of mechanical force’.

0379-0738 / 00 / $ – see front matter  2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0379-0738( 00 )00269-3
154 T. Ohshima / Forensic Science International 113 (2000) 153 – 164

In forensic pathology, wounds are classified into al hemorrhage such as subarachnoid hemorrhage or a
three different types according to the manner of the vessel lesion like coronary artery occlusion [4–12].
mechanical force to the skin as follows: blunt injury, With the advance of clinical diagnostic devices,
sharp weapon injury and gunshot wound. Further- MRI examination is applied to forensic autopsy
more, blunt injuries are sub-classified into abrasion, [13,14]. In MRI examinations, sagittal views as well
laceration, bruise and intradermal bleeding. Sharp as horizontal ones can be obtained, and this is the
weapon injuries are also sub-classified into incised most advantage in MRI examinations.
wound and stab wound.
3.2. Endoscopic examination
2.2. The purposes of wound examination
Endoscopic instruments are now used in forensic
The purposes of wound examination are described autopsy [15,16]. The most advantage of endoscopic
in Gradwohl’s textbook [2] as follows: (1) whether it examination is that the cavities and sinuses can be
was caused before, at the time of, or after death; (2) inspected and photographed without their mor-
how it was caused; (3) what caused it; (4) what phological damage. Especially, Amberg et al. [16]
amount of force was required or produce it; (5) what recommended the endoscopic inspection of the eye
degree of injury has resulted from it and whether it ground, the external auditory meatus up to tympanic
has influenced death or caused disability. Generally, membrane and the naso- and laryngopharynx. Fur-
wound vitality, the characteristics of mechanical thermore, Slaughter and Brown [17,18] used a
force, and the causal relation between the mechanical colposcope for examination of genital injuries in rape
force and the person’s death or disability are the victims and emphasized that the usage of colposcope
major points to be elucidated. significantly increased the reliability of identifying
genital injuries in those cases.

3.3. Diphanoscopic examination

3. The application of clinical techniques and
devices to wound examination It is sometimes difficult to detect subcutaneous
hematomas with the naked eye. Naturally, in autopsy
Although macroscopic and histological examina- cases, such hematoma can be easily detected with a
tions are routinely performed at forensic autopsy, cutaneous incision. However, in the cases of living
wound examination should be improved and reno- persons, it is impossible to find subcutaneous hema-
vated with the advance of medical science and toma by skin incision. Horisberger and Krompecher
medical appliances. Thus, it is desirable that modern [19] reported that forensic diaphanoscopic examina-
diagnostic techniques and devices are used at foren- tion was a highly sensitive and specific procedure to
sic practices. determine the presence or absence of subcutaneous
hematomas.
3.1. Radiological examination including magnetic
resonance imaging 3.4. The application of operation microscopes to
forensic practice
Schmidt and Kallieris [3] reported that, at forensic
autopsy, radiological examination was very useful In 1979, Holczabek and Depastas [20] reported on
for the identification of foreign bodies, air embolism, the application of operation microscope to forensic
bone fracture, and other internal abnormalities before diagnosis and stated that an operation microscope
dissection. Especially, in the case where a bullet has should be employed as much as possible, because of
still remained in a deceased body, radiological practical and scientific reasons. Moreover, Monahan
examination much contributes to confirm the location and Harding [21] performed the experimental study
of the bullet. Moreover, postmortem angiography is on damage to clothing. Their study with an operation
also performed to determine an origin of intracerebr- microscope revealed that it is possible to discrimi-
 T. Ohshima / Forensic Science International 113 (2000) 153 – 164 155

nate whether a damage to clothing was caused by a video-records were simultaneously obtained from the
single-edge or double-edge knife. monitor (Fig. 1). Therefore, this system presents the
At our department, wound examination with a advantages on the occasion of instruction and train-
binocular operation microscope has been routinely ing of young forensic pathologists and clinical
performed for close observation of skin wounds (Fig. physicians as well as police officers.
1) [22]. In abrasion, small epidermal tags, which
became a key for the determination of external force
direction, were clearly observed. In laceration, crush- 4. Examination of gunshot wounds
ing of the epidermis was observed at the wound
margin, and continuous bridges of connective tissue Karger et al. [23] mentioned that gunshot wounds
and / or blood vessels were also found within the should be examined radiologically, pathologically
wound cavity. In contrast, in incised wound, the and histologically. Especially, it is important to
epidermis was, in principle, intact up to the wound estimate firing range and to differentiate the entrance
margin, and there was no continuous bridge within from the exit. Brown and co-workers [24,25] per-
the wound cavity. According to these differences, formed an image analysis of gunshot residue on
therefore, it was possible to distinguish laceration entry wounds and concluded that it is possible to
from incised wound. Ligature mark due to hanging distinguish a contact shot from all other more distant
was clearly observed, and, additionally, petechial shots, and shots at distance of #20 cm from those at
hemorrhages, as vital reaction, were recognized distances of .20 cm. The image analysis is applic-
within the mark (Fig. 2). In stab wounds caused by a able to the cases where the distinction between a
single-edge knife, morphological difference between closer or a more distant range is the main issue.
the two ends made by the sharp blade or the blunt According to the article by Ohtsuji et al. [26], as one
side was recognized. Such abnormal lesions of blood of the methods for the discrimination of entrance
vessels as mechanical tear of the vertebral artery (the from exit wound, the analysis of inorganic lead in
origin of subarachnoid hemorrhage) and atheros- wound specimens using atomic absorption spec-
clerotic stenosis of the coronary artery were detect- trophotometer is useful, and inorganic lead con-
able. Microscopic image was visualized on a closed- centration in the entrance wound is much higher than
circuit television monitor, and color photographs and that in the exit one.

Fig. 1. The operation microscope system at my department.

156 T. Ohshima / Forensic Science International 113 (2000) 153 – 164

Fig. 2. Ligature mark. (a) Macroscopic view of the ligature mark. (b) Microscopic view of the ligature mark. Arrows indicate petechial
hemorrhage with in the mark.

5. The determination of wound vitality or estimation of bruises is important when a cadaver or

wound age injured person has plural bruises of different ages,
especially in child abuse cases. Forensic pathologists
It is also necessary to determine whether a wound can determine how long a victim has been abused by
has vital reaction, or, if the wound has vital reaction, the age estimation of bruises.
how long before the wound has been sustained. In Of course, in open wounds such as stab wounds,
other words, the determination of wound vitality or incised wounds and lacerations, the determination of
wound age has to be always performed. For example, wound vitality or wound age is required. For this
age of bruises are estimated based on the temporal aim, pathophysiology of skin wound healing is to be
change or transition of their color [27–29]. Such age understood thoroughly. Generally, skin wound heal-
 T. Ohshima / Forensic Science International 113 (2000) 153 – 164 157

ing process is composed of three different phases enzyme-histochemical method [30–37], HPLC
like inflammatory, proliferative and maturation (high-performance liquid chromatography) [54,55],
phases. In each phase, various kinds of biological electrofocussing [56,57] and enzymological [68,59]
substances are closely involved. Examination of the method. Berg and co-workers [60–63] examined
dynamics of such biological substances becomes a serotonin, histamine and histidine-decarboxylase ac-
clue to find an available marker for wound vitality or tivity in skin wound. Laiho [64] examined the
wound age. activity of myeloperoxidase in skin excoriations or
incisions, and he also examined the influences of
5.1. Previous studies on the determination of blood loss, wound depth, alcohol and some
wound vitality or wound age determination medicines to the activity of myeloperoxidase [65,66].
Eisenmenger et al. [67] investigated the expression
At first, Raekallio and co-workers [30–37], as a of collagens in during skin wound healing, and
pioneer of this research field, examined the enzyme Oehmichen and co-workers [68,69] examined RNA
activity at the zones of a wound using enzyme- and DNA synthesis of epidermal basal cells after
histochemical method. A portion of up to 500 mm wounding. Recently, for more objective evaluation of
from the wound edge was named the central zone, histological findings, morphometry, together with
and, a portion of up to 100–200 mm from the central immunohistochemistry or histochemistry, has been
zone was called the peripheral zone. Raekallio’s performed [70]. Dreßler et al. examined adhesion
studies revealed that the enzyme activity decreased at molecules (ICAM [71] and VCAM [72]) and selec-
the central zone. On the other hand, at the peripheral tin [73]. Apoptosis during wound healing is now
zone, an increase of enzyme activity was observed. focused, and in situ TUNEL method [74] and p53
At the peripheral zone of wound, enzymatic activity products [75] are also applied to wound age de-
was observed earlier than histological findings like termination.
inflammatory cell infiltration, and, especially, adeno- In addition, there are forensic studies on the age
sine triphosphatase was earliest activated at 1 h after determination of brain injury as well. Oehmichen
wounding, followed by esterase, aminopeptidase, and co-workers [76–78] performed systematic his-
acidphosphatase and alkalinephosphatase. tological examinations on brain injuries with various
Betz and co-workers [38–49] performed a series postinfliction intervals. Hausmann et al. [79] ex-
of immunohistochemical studies on wound age de- amined immunohistochemically the surface antigens
termination. It is concluded that, among these param- of neutrophils and macrophages in brain injuries.
eters, P-selectin is the earliest marker, followed by
fibronectin, E-selectin and ICAM-1, and that ex- 5.2. The dynamics of cytokines in skin wound
tracellular matrices such as collagens, tenascin and healing
laminin become useful parameters at 1–5 days after
injury [50]. Furthermore, subtyping of macrophages At our department, the dynamics of cytokines
is also useful for wound age determination in the during skin wound healing has been mainly investi-
later phase of wound healing process [51]. gated from the viewpoints of wound age determi-
In addition to the studies by Raekallio and Betz, nation. Cytokine is glycoprotein produced by various
there are lots of findings by our senior colleagues on kinds of cells such as neutrophils, lymphocytes,
the determination of wound vitality or wound age. macrophages, etc. Cytokines have multi-biological
Janssen [52] comprehensively reviewed vital re- functions involved in immune, hematopoietic, endo-
actions in his famous monograph. Walcher [53] crine, nerve and inflammation systems. At present, it
attempted wound age determination using has been well-known that cytokines have a close
hemosiderin deposits detected by Prussian blue relation with wound healing process. In particular,
staining, and he reported that hemosiderin deposits such cytokines as interleukin (IL)-1, IL-6 and tumor
were detectable at the ninth day after injury. There- necrosis factor-a (TNFa) are very important chemi-
after, various kinds of methods were employed for cal mediators in the acute inflammatory reaction.
the determination of wound vitality or wound age; Experimentally, the dynamics of such inflamma-
158 T. Ohshima / Forensic Science International 113 (2000) 153 – 164

tory cytokines as IL-1a, IL-1b, IL-6 and TNFa common. However, there was no study using mRNA
during mouse skin wound healing was examined analysis for forensic wound examination, and the
using enzyme-linked immunosorbent assay and im- affect of postmortem changes on mRNA left un-
munostaining [80]. Keratinocytes of the mouse skin known. Therefore, mRNA as well as protein of
were immunostained for uninjured skin specimens cytokines has been examined, and its applicability to
with antibody against each cytokine. In the wounds forensic practice is discussed.
aged 3–12 h, the source of these cytokines was Temporal expression of mRNA for the inflamma-
neutrophils (Fig. 3). Thereafter, with an increase of tory cytokines such as IL-1a, IL-1b, IL-6 and TNFa
postinfliction interval, macrophages and fibroblasts during mouse skin wound healing was examined
showed positive reactions for each cytokine. The using the reverse transcriptase-polymerase chain
levels of TNFa and IL-1b began to rapidly elevate reaction (RT-PCR) and in situ hybridization tech-
and reached a peak at 3 h after wounding (Fig. 4a). niques [81]. After incision, an increase in each
IL-1a and IL-6 reached a peak at 6 and at 12 h, cytokine mRNA level was observed by RT-PCR.
respectively. In addition, ‘rebound’ of every cytokine The IL-6 mRNA level peaked at 6 h, while the peak
level was observed at 72 h after wounding (Fig. 4b). levels of mRNA for IL-1a, IL-1b and TNFa
These results indicated that these cytokines have a occurred between 48 and 72 h. All cytokine mRNA
close relation with wound remodeling as well as levels were almost normalized after 240 h. In the
inflammation. From the viewpoint of forensic pathol- early phase of wound healing, infiltrating polymor-
ogy as well, these inflammatory cytokines show the phonuclear neutrophils were labeled with antigens
possibility of available markers for wound age probes for IL-1a, IL-1b and TNFa mRNA (Fig. 5a).
determination Thereafter, infiltrating mononuclear cells and spin-
dle-shaped fibroblastic cells showed positive signals
5.3. The mRNA expression of inflammatory for all the cytokines examined (Fig. 5b,c). These
cytokines in skin wound healing findings indicate that IL-1, IL-6, and TNFa are
involved in wound healing. From the viewpoint of
Nowadays, medical research at molecular level are forensic pathology, the temporal characteristics of

Fig. 3. Arrow heads indicate positive immunoreaction for IL-1a in the cytoplasm of some neutrophils at 3 h after mouse skin incision. As
for the other cytokine such as IL-1b, IL-6 and TNFa, similar findings were observed.
 T. Ohshima / Forensic Science International 113 (2000) 153 – 164 159

Fig. 4. Time course of inflammatory cytokine level during mouse skin wound healing: (a) 0–24 h after incision, (b) 0–240 h after incision.

the cytokine mRNA expression may have a potential to 180 min [82]. The amount of IL-10 mRNA in
to indicate wound age. each skin specimen was evaluated using RT-PCR. A
significant increase in IL-10 mRNA occurred be-
5.4. The mRNA expression of inhibitory cytokine tween 30 and 180 min after incision. During the
( IL-10) in mouse skin wound 5-day postmortem interval, the increase in time-
dependent IL-10 mRNA expression was maintained,
The expression of IL-10 mRNA was examined and no significant increase in IL-10 mRNA expres-
with mouse incision with postinfliction interval of 0 sion occurred in the postmortem control (Fig. 6).
160 T. Ohshima / Forensic Science International 113 (2000) 153 – 164

Fig. 5. These photographs show the localization of IL-1b mRNA during mouse skin wound healing. (a) At 12 h after wounding, neutrophils
(arrows) expressed IL-1b mRNA. (b) Three-day-old wound. Arrows indicate infiltrating mononuclear cells showing positive signals for
IL-1b mRNA. (c) In 6-day-old wound, spindle-shaped fibroblastic cells (arrows) were labeled with IL-1b antisense probe. Similar results
were obtained for mRNA of IL-1a, IL-6 and TNFa.
 T. Ohshima / Forensic Science International 113 (2000) 153 – 164 161

Fig. 6. This figure shows how postmortem interval influenced the expression of IL-10 mRNA and b-actin mRNA. Time-dependent IL-10
mRNA expression was maintained for 5 days postmortem.

The increased expression of IL-10 mRNA could be more objective evaluation of immunohistochemical
considered a vital reaction in skin specimens. This findings obtained, the ratio of number of IL-1a-
study demonstrated the possible use of mRNA positive infiltrating cells to the total number of
analysis for forensic wound examination, since infiltrating cells was evaluated. A considerable in-
mRNA was detectable by RT-PCR over a longer crease in the IL-1a-positive cell ratio was observed
postmortem time course than expected. in wound specimens aged 4 h to 1 day. The ratio of
IL-1a-positive cells gradually decreased in wounds
5.5. Practical application of IL-1a to wound age aged between 1.5 and 21 days. Statistically, a
determination significant difference in the mean ratio of IL-1a-
positive cells was observed between wound speci-
Practical possibility of IL-1 a as a marker for mens with wound ages ranging from 4 h to 1 day
wound age determination was examined using (32.869.7%) and those aged over 1.5 days
human skin wounds [83]. Immunohistochemically, (17.567.2%). These results show that IL-1a-positive
IL-1a-positive cells in human skin wounds were ratio, considerably exceeding 30%, indicate a post-
consistent with those in mouse skin wounds. For infliction interval of 1 day or less.
162 T. Ohshima / Forensic Science International 113 (2000) 153 – 164

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