Forensic science international quantitative analysis of proinflammatory cytokines (IL 1β, IL 6, TNF

Forensic Science International
113 (2000) 251–264

www.elsevier.com / locate / forsciint

Quantitative analysis of proinflammatory cytokines
(IL-1b, IL-6, TNF-a) in human skin wounds
a,
b
a
W. Grellner *, T. Georg , J. Wilske
a

Institute of Forensic Medicine, Saarland University, Building 42, D-66421 Homburg /Saar, Germany
b
Institute of Medical Biometrics, Saarland University, Homburg /Saar, Germany

Abstract
Proinflammatory cytokines play an important role in the mediation of inflammation and trauma. They could be useful for
the determination of vitality and wound age. In the present study, 144 human skin wounds due to sharp force were
investigated. The material was collected during operations (N596) and postmortem examinations (N548). The wound age
varied from several seconds or minutes to 9 days. Control skin was available in each individual. The tissue specimens were
homogenized and extracted in a solution of PBS and protease inhibitors. Interleukin-1b (IL-1b), interleukin-6 (IL-6) and
tumour necrosis factor alpha (TNF-a) were measured by quantitative ELISA analysis. Statistical evaluation was performed
by the t-test using the quotients of levels (wound sample / control skin). In surgical specimens the cytokine levels revealed a
clear tendency to increase with wound age. IL-1b in early skin wounds (#30 min) and TNF-a after a wound age of 1–2 h
demonstrated statistically significant changes in comparison with control skin (P,0.05). In autopsy samples with severe
traumatization excessive elevation of cytokine levels was observed: IL-1b, IL-6 and TNF-a showed significant increases
(P,0.001–0.05) in stab and incised wounds with very short survival times of less than 5 min, but not in possibly supravital
injuries. Elevated IL-6 levels persisted in older wounds (.24 h, P,0.05). The quantitative analysis of proinflammatory
cytokines in wound extracts can contribute to the determination of vitality and wound age, in particular in the very early

post-traumatic interval (classic stab wounds).  2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Proinflammatory cytokines; Interleukin-1b; Interleukin-6; Tumour necrosis factor alpha; Wound age; Vitality

1. Introduction
Cytokines are mediators with multiple functions,
including the initiation or influence of numerous
biological processes such as inflammation, sepsis and
wound healing [1–9]. The proinflammatory cytokines interleukin-1b (IL-1b), interleukin-6 (IL-6)
and tumour necrosis factor-a (TNF-a) play key roles
within the cytokine network as it is frequently called
(see reviews in Refs. [10–13]). This term refers to
*Corresponding author. Fax: 149-6841-166-314.

the close relationship between various cytokines and
their pathways. Minute concentrations, rapid stimulation and short durations of activity characterize the
autocrine / paracrine regulation of these peptides.
They are produced by a variety of cell types such as
macrophages, thrombocytes and keratinocytes. The
features mentioned could make cytokines to an
appropriate tool for the evaluation of vitality and
early wound age.
In the present study, the proinflammatory mediators IL-1b, IL-6 and TNF-a were determined
quantitatively in extracts of human skin wounds and

0379-0738 / 00 / $ – see front matter  2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0379-0738( 00 )00218-8


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W. Grellner et al. / Forensic Science International 113 (2000) 251 – 264

tested for their usefulness in the estimation of vitality
and wound age.

2. Material and methods

2.1. Material
In the present study, 144 human skin wounds due
to sharp force were investigated. The material was
collected during operations (N596) and postmortem
examinations (N548). Control skin from uninjured
areas was available in each individual.

2.1.1. Surgical material
A total of 96 wound samples with control skin
were collected; 54 from males and 42 from females,
average age: 56.3615.4 years. In dependence on
weight and volume, the tissue could be used for the
determination of one or more markers and was
distributed as follows: IL-1b (N543), IL-6 (N549)
and TNF-a (N546).
The wound age varied from 7 min to 5.5 h with
special emphasis on the early post-traumatic interval
up to 3 h (average: 73656 min). Four wound age
classes were formed: #30 min (N523), 31–60 min
(N531), 61–120 min (N523) and .120 min (N5
19). Most of the skin samples originated from the
abdominal region (N584).
2.1.2. Autopsy material

A total of 48 wound samples with control skin
were collected; 33 from males and 15 from females,
average age: 45.8616.4 years. Because of the higher
tissue weight, three markers could be determinded in
most of the samples: IL-1b (N548), IL-6 (N547)
and TNF-a (N544).
The wound age varied from several seconds or
minutes in homicides due to sharp force and injuries
from railway suicides to a maximum of 9 days in
surgically treated wounds (average: 23.2651.2 h).
Three wound age classes were formed: # ca. 5 min
(N532), 1–24 h (N56) and .24 h (N510). In the
first group the survival time could not exactly be
defined, as there were ‘‘classic’’ homicides and
suicides by stab and incised wounds where the
involved persons were already found dead. Due to
the presence of severe injuries of greater blood

vessels the above mentioned time interval of ‘‘up to
several min’’ (#5 min) was concluded as the probable wound age. In this group, there were also cases
(railway suicides, emergency measurements in the
agonal or early supravital period) with extremely
short survival times or possibly supravital injury.
This small subgroup (N512) was additionally evaluated separately.
Most of the skin samples originated from the
abdominal region (ca. 25%), but other anatomical
locations were also present (extremities, thorax). The
postmortem interval was between 6 h and 20 days
(average: 74687 h).
Individuals with known immunodeficiencies or

immunotherapy were excluded from the study in
both surgical and autopsy samples.
The wound specimens were taken parallel to the
wound margin. They measured approximately 0.5–
1.0 cm in length and 0.2–0.3 cm in width. The
subcutis was removed. The skin samples were shock
frozen in liquid nitrogen and could be stored at
2708C until analysis. The average weight was
62632 mg in surgical samples and 84619 mg in
postmortem specimens, respectively.

2.2. Tissue extraction
The complete extraction was performed under
cooling conditions (#48C, in liquid nitrogen, on ice)
because of the known susceptibility of cytokines. We
developed our own protocol according to procedures
for the quantitation of stem cell factor (SCF) from
placental tissue [14] and IL-8 from psoriatic skin
[15].
In brief, the frozen skin specimens were homogenized mechanically and by incubation in an oscillating
mill under the permanent addition of liquid nitrogen
(1–2 min). The resulting product, which resembled a
powder, was transferred to cups, weighed (wet
weight) and extracted in a ten-fold volume of
phosphate buffered saline (PBS, pH 7.4) and protease inhibitors [2 mM phenylmethylsulfonyl fluoride
(PMSF);
5
mM
ethylenediaminetetraacetate
(EDTA); 1 mg / ml of leupeptin, antipain, aprotinin

and pepstatin A, respectively]. The extraction took 1
h at 48C under permanent agitation. Then the samples were centrifuged for 20 min at 15 000 g and


W. Grellner et al. / Forensic Science International 113 (2000) 251 – 264

48C. The resulting solution (soluble cytokine fraction) could be stored at 2708C until ELISA analysis.
In some samples, the sediments were re-extracted
overnight in a solution of 8 M guanidinium, pH 7.4,
and the protease inhibitors were, as mentioned
above, dialyzed for 8 h to remove the guanidinium
and finally centrifuged again. The resulting solution
contained the matrix associated cytokine fraction.

253

P,0.05 were regarded as statistically significant.
The arithmetical means and the standard deviations
were calculated. The chi-square test and the correlations of Kendall and Spearman were used for testing
the results on independence of age, gender, anatomical site and postmortem interval.

3. Results

2.3. Determination of protein content
3.1. General remarks
In some samples the protein content (mg / ml) was
determined by three-fold photometric measurements
at 560 nm according to the microtiter plate method
of the bicinchoninic acid (BCA) protein assay
(Pierce, USA) [16–19].

2.4. ELISA
Interleukin-1b (IL-1b), interleukin-6 (IL-6) and
tumour necrosis factor alpha (TNF-a) were measured
by quantitative ELISA analysis (kits and protocols of
BioSource, USA) using a microtiter plate reader
(photometry at 450 nm) and an automatic calculation
of results. The respective cytokine levels were
determined in wound samples and control specimens
by means of double measurements requiring 100–
200 ml of extract. The results had the dimension of
pg / ml. The detection limits were 1 pg / ml for IL-1b,
2 pg / ml for IL-6 and ,1 pg / ml for TNF-a, respectively.

2.5. Evaluation and statistical analysis
The following parameters were considered: age,
gender, wound age, postmortem interval in autopsy
cases, anatomical site of wounds, sample weight,
protein content and ELISA results.
The following quotients were calculated and tested: pg of cytokine / mg of protein, pg of cytokine / g
of tissue and pg of cytokine / mg of protein / g of
tissue.
Testing all parameters, the ELISA results of the
PBS extracts in pg / ml were finally related to the wet
weight of skin samples (pg / g). These values were
independent of artificial changes. Statistical evaluation was performed by the t-test using the quotients
of levels (wound sample / control skin). Values of

The results of the quantitative analyses differed
considerably between the wound samples from the

surgical procedures and those from the postmortem
examinations. Both parts are therefore depicted
separately (Sections 3.2 and 3.3).
Common characteristics of this study were as
follows: the decisive variable was the content of
soluble cytokines as extracted by PBS. It did not
disappear in a possibly greater pool of matrix
associated cytokines as extracted by guanidinium. It
had to be related to the weight of skin samples
(pg / g). These results showed great interindividual
variability of cytokine levels concerning also the
normal values in the control skin (Table 1). Therefore, it was in addition necessary to calculate the
quotients of levels (wound sample / control skin),
other correlations or quotients did not give useful
results. Using this method, a good correlation between the quantitative results and the wound age was
possible. The results were statistically independent of
age, gender, anatomical site or postmortem interval.
The partially performed determination of the
protein content revealed the following values which
were not used in the statistical evaluation:
• surgical material: 1.4860.44 mg / ml in skin
wounds (N578) and 1.3560.47 mg / ml in control
skin (N574)
Table 1
Cytokine levels in normal human skin
Cytokine

Level in normal skin a
(vital, pg / g)

Level in normal skin a
(postmortem, pg / g)

IL-1b
IL-6
TNF-a

127.56127.6
205.16159.0
1470.863186.6

329.66570.8
190.26384.3
627.161503.6

a

Mean values and standard deviation.


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W. Grellner et al. / Forensic Science International 113 (2000) 251 – 264

• autopsy material: 1.9560.60 mg / ml in skin
wounds (N529) and 1.5360.47 mg / ml in control
skin (N529).

3.2. Wound samples from surgical procedures
In surgical specimens the cytokine levels revealed

a clear tendency to increase with wound age. In this
group IL-1b and TNF-a proved to be the most
valuable markers.

3.2.1. Interleukin-1b
The quotients of IL-1b varied from 0.41 to 3.44
with increasing tendency in wounds with an age of
hours (Fig. 1).
When compared to normal skin, IL-1b showed a
statistically significant decrease in the first wound
age class (#30 min) with an average quotient of
0.7960.28 (P,0.05). The quotients exhibited then
an almost linear increase with values of about 1.5 in
wounds with an age of .2 h (quotient: 1.5360.76,
P50.056) (Fig. 2, Table 2).
3.2.2. Interleukin-6
In comparison with IL-1b, the quotients of IL-6

demonstrated a considerably greater variability with
values from 0.23 to 11.26. They also increased with
higher wound age (Fig. 3).
The quotients amounted to approximately 1.0 (i.e.,
identical to control skin) in wounds aging up to 1 h
and doubled in the following wound age classes
(61–120 min: 1.9562.49; .120 min: 2.0863.26)
(Fig. 4). However, as shown by the standard deviation, the variability was also considerable in posttraumatic intervals of more than 1 h, so that the
increase was not significant (Table 2).

3.2.3. Tumour necrosis factor-alpha
The quotients of TNF-a were mostly greater than

1.0, the variation was between 0.37 and 8.43 (Fig.
5). An increase with higher wound age was not
observed, but TNF-a was the only cytokine with an
average quotient of 1.8461.57 for all skin wounds
(independent of wound age) which demonstrated
high significance compared to control skin (P5
0.001).
The various wound classes are shown in Fig. 6:
the TNF-a quotients elevated to 1.6161.20 already
in the early post-traumatic interval (#30 min, P5
0.066). Apart from a statistically significant decrease

Fig. 1. Distribution of IL-1b quotients in human skin wounds (surgical material).


W. Grellner et al. / Forensic Science International 113 (2000) 251 – 264

255

Fig. 2. IL-1b in human skin wounds (surgical material). Mean quotients and standard deviations.

in the class from 61 to 120 min (quotient:
1.3960.54, P,0.05) the quotients remained over 2
(Fig. 6, Table 2).

average of 1.7260.92 in the shortly survived stab
and incised wounds of homicides and suicides was
highly significant (P,0.001). IL-1b fell back to
values in normal skin in the medium wound age
group from 1 to 24 h (quotient: 1.2960.65) and

disclosed the highest levels in the later wound age
phase .24 h (quotient: 3.2364.87). Due to the high
standard deviation statistical significance could not
be reached.

3.3. Wound samples from autopsies
In autopsy samples with severe traumatization, an
excessive elevation of cytokine levels was frequently
observed exceeding the results of surgical samples
by far. IL-6 proved to be the ‘‘key marker’’.

3.3.2. Interleukin-6
The quotients of IL-6 demonstrated again – comparable to surgical wounds – the greatest range with
values between 0.23 and 80.82. The maximum
average IL-6 levels were detectable in the medium

3.3.1. Interleukin-1b
The quotients of IL-1b ranged from 0.42 to 16.84
with an increasing tendency in higher wound age
.24 h (Fig. 7, Table 3). The rapid increase to an

Table 2
Proinflammatory cytokines: statistical significances for surgical samples a
Wound age

IL-1b
IL-6
TNF-a
a

#30 min

31–60 min

.120 min

61–120 min

P

N

P

N

P

N

P

N

,0.05
n.s.
0.066

9
11

15

n.s
n.s.
0.061

9
11
13

n.s
n.s.
,0.05

15
17
11

0.056
n.s.
n.s.

10
10
7

P-values of quotients, n.s.5not significant.


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W. Grellner et al. / Forensic Science International 113 (2000) 251 – 264

Fig. 3. Distribution of IL-6 quotients in human skin wounds (surgical material).

Fig. 4. IL-6 in human skin wounds (surgical material). Mean quotients and standard deviations.


W. Grellner et al. / Forensic Science International 113 (2000) 251 – 264

Fig. 5. Distribution of TNF-a quotients in human skin wounds (surgical material).

Fig. 6. TNF-a in human skin wounds (surgical material). Mean quotients and standard deviations.

257


W. Grellner et al. / Forensic Science International 113 (2000) 251 – 264

258

Fig. 7. IL-1b in human skin wounds (autopsy material). Mean quotients and standard deviations.

wound age class from 1 to 24 h (Fig. 8, Table 3).
Despite considerable variability, the increase of IL-6
in stab wounds from homicides and suicides with
extremely short survival times was so excessive that
it proved to be statistically significant (quotient:
7.08615.73, P,0.05). In one case (stab wound) an
over 80-fold increase of IL-6 was measured compared to normal skin. Despite even greater average

quotients (9.21611.42) in the wound age class from
1 to 24 h, the high standard deviation and the
relatively small case number prevented statistical
significance. However, statistically significant results
were again found in ‘‘older’’ skin wounds .24 h
(quotient: 5.1064.34, P,0.05).
Table 3
Proinflammatory cytokines: statistical significances for autopsy
samples a
Wound age

IL-1b
IL-6
TNF-a
a

#5 min

.24 h

1–24 h

P

N

P

N

P

N

,0.001
,0.05
,0.01

32
32
29

n.s.
n.s.
n.s.

6
5
6

n.s.
,0.05
n.s.

10
10
9

P-values of quotients, n.s.5not significant.

3.3.3. Tumour necrosis factor-alpha
TNF-a revealed the maximum quotients similar to
IL-6 in the medium wound age interval from 1 to 24
h (Fig. 9, Table 3). The values for all skin wounds
varied from 0.67 to 10.05. Among the proinflammatory cytokines, TNF-a showed the absolute lowest
but most constant increase in stab wounds with very
short survival times so that this marker was markedly
elevated in the earliest post-traumatic interval (quotient: 1.5560.99, P,0.01). The medium wound age
class from 1 to 24 h was characterized by greater
absolute increases, but also by higher variations, so
that statistical significance could not be demonstrated
(quotient: 3.5963.41). TNF-a fell back to normal
levels similar to the control skin in the late wound
age group (quotient: 1.0460.33).

3.3.4. Final summary of postmortem investigations
The quantitative analysis of proinflammatory cytokines demonstrated its advantages especially in the
very early post-traumatic interval of less than 5 min
(vitality) (Fig. 10, Table 3). All three parameters
IL-1b, IL-6 and TNF-a showed significant increases
(P,0.001–0.05) in stab and incised wounds despite


W. Grellner et al. / Forensic Science International 113 (2000) 251 – 264

Fig. 8. IL-6 in human skin wounds (autopsy material). Mean quotients and standard deviations.

Fig. 9. TNF-a in human skin wounds (autopsy material). Mean quotients and standard deviations.

259

W. Grellner et al. / Forensic Science International 113 (2000) 251 – 264

260

Fig. 10. Summary of proinflammatory cytokines in human skin wounds (autopsy material). Mean quotients and significances.

the shortest survival times (homicides and suicides
by sharp force with rapid exsanguination).
Separate evaluation of the subgroup (N512) with
extremely short survival times (railway suicides) or
possibly supravital injuries (emergency measurements) exhibited only slightly elevated cytokine
quotients (IL-1b: 1.5060.89; IL-6: 1.2960.57; TNFa: 1.5761.04). Statistically significant differences to
the control skin were not found.
The medium post-traumatic interval from 1 to 24 h
was characterized by high levels of IL-6 and TNF-a.
Significant results were not demonstrable.
High and statistically significant IL-6 levels persisted in older wounds (.24 h) and also IL-1b
remained elevated, whereas TNF-a fell back to basal
values (Fig. 10).

4. Discussion
After a traumatic event cytokines are primarily
synthesized and / or released at the site of tissue
destruction (autocrine / paracrine effect). Systemic
levels follow in the later course of an inflammatory

or traumatic process. Therefore, it made sense to
quantitate the cytokine response directly in the skin

wound. Despite vast literature data on cytokines in
general, there are only a few comparable studies
using this method. The main problem is the establishment of an efficient extraction. Cytokines act in
minimal concentrations and are relatively unstable,
so that it may be difficult to get quantities above the
detection limit of ELISAs. In addition, skin is an
especially resistant material.
In our study, we used an intensive mechanical
homogenization and an extraction in a solution of
PBS and protease inhibitors. By this way it was
possible to obtain the pool of soluble cytokines
within the dermal cells which is subject to rapid
alterations after stimulation. In order to estimate the
relation between the soluble and the matrix associated cytokine pool, we firstly also used a re-extraction by guanidinium. The results, however,
showed that the soluble pool was much greater in
most of the cases (relation: 0.6–5.8) and that the
re-extraction had no additional advantage. Slight
variations in the extraction yield did not influence the
final result, as the control skin of the same individual
was measured in the same examination and the


W. Grellner et al. / Forensic Science International 113 (2000) 251 – 264

quotients between wound levels and levels in control
skin were used. The absolute cytokine concentrations
did not allow an evaluation of the wound age, as the
interindividual variation was considerable. It was
useful to relate the concentrations to the skin weight,
an additional consideration of the protein content like

Kondo and Ohshima [20] had no advantage.
One revealing result of our study was the marked
graduation of the cytokine reactivity between surgical and autopsy material. These differences were
obviously due to the considerably more severe
traumatization in autoptic skin wounds (stab wounds
of homicides) with additional manifestation of
hemorrhagic shock. By contrast, during operations,
preservation of the tissue and avoidance of severe
laceration is attempted.
In the surgical skin wounds, up to 11-fold increases (IL-6) could be observed (IL-1b: up to
3.5-fold; TNF-a: up to 8.5-fold). On the whole, the
levels of proinflammatory cytokines increased with
higher wound age. In comparison with normal skin
significant differences, however, were only found for
IL-1b in the early post-traumatic interval (#30 min)
and for TNF-a in the wound age class from 1 to 2 h.
On an average the levels doubled in the various
wound age groups when compared to controls.
In skin wounds collected at autopsies, much
higher levels were detected. Frequently excessive
increases were observed even in (stab) wounds with
very short survival times. The postmortem ‘‘key
marker’’ IL-6 exhibited up to 81-fold increases (IL1b: up to 17-fold; TNF-a: up to 10-fold). All three
parameters demonstrated statistically significant elevations of levels in the very early wound age group
up to 5 min. In the case of positive reactions the
proinflammatory cytokines can be regarded as very
useful vitality markers for stab and incised wounds.
This is supported by the fact that wounds with
extremely short survival times did not disclose
significant changes of cytokine content. Thus, these

markers are characterized by a good discriminating
function between vital and supravital injuries (good
specificity). On the other hand a negative result in
the analysis of cytokines can not exclude vitality
(poorer sensitivity).
Former quantitative studies on cytokines were
mainly conducted by means of blood / serum levels
and cell cultures or animal experiments [21–34]. To
our knowledge extracts of human organs have only

261

been used for the determination of IL-8 (skin) [15]
and stem cell factor (placenta) [14].
Furthermore, Kondo and Ohshima [20] presented
the single study under medicolegal aspects: in a
murine animal model experimentally-incised wounds
were examined quantitatively for IL-1a, IL-1b, IL-6
and TNF-a. Their data from a controlled animal
experiment with maximum average cytokine quotients of approximately 2 resemble our results in
surgical human wounds. However, increases were
observed much later than in our study and statistical
significances were not mentioned. Kondo and Ohshima observed maximum levels for IL-1b and TNFa not before 3 h and for IL-6 even not before 12 h
after wounding. This is obviously not in concordance
with the forensic reality, since massive increases can
be detected in severely traumatized tissues of
homicide victims even after very short survival
times. Thus, the local cytokine analysis at the wound
site can be quite useful, at least as far as macroscopically ‘‘fresh’’ skin is available after postmortem
intervals of up to approximately 1 week.

There are various reasons for the rapid increase of
proinflammatory factors. In the first place local
release from preformed stores comes into question. It
is known that in particular multipotent keratinocytes,
mast cells, sweat glands and partly macrophages
contain higher amounts of various cytokines (e.g.
[21,35–40]). Numerous mediators are stored there as
inactive precursors or in the active form [37,41,42].
The influence of the severe traumatization with
finally fatal outcome must not be underestimated as
well: it leads to a considerable activation of the
cytokine cascade exceeding other stimuli in quantity
and kinetic by far. In massive trauma factors such as
hemorrhagic shock with systemic elevation of proinflammatory cytokines, local ischemia and hypoxia
become relevant, inducing the activation of cytokines
[43,44].
In addition, the transudation of the tissue with
blood components (soluble cytokines in the serum,
additional release from cellular elements such as
monocytes) may contribute to the rapid quantitative
increase of proinflammatory mediators as well. However, the negative results of possibly supravital
injuries point to the fact that intact circulation is
required and that sole passive transudation (e.g.,
postmortem injury) is not sufficient.
It seems to be evident that all these factors lead to


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W. Grellner et al. / Forensic Science International 113 (2000) 251 – 264

an earlier positive reaction in a quantitative biochemical cytokine analysis than in an immunohistochemical method where mainly cytokines bound
on the cell surface are detected. The time intervals
for the earliest reactions as stated in this study are
consequently markedly smaller than the comparable
reaction times observed in immunohistochemistry
(own data, publication in preparation).
Our findings are in concordance with the results of
the clinical and experimental literature on the role of
proinflammatory factors in trauma and inflammation
or sepsis. The serum levels of TNF-a, IL-1a and
IL-6 increase after operation and trauma [3]. IL-1a,
IL-1b, IL-6, TNF-a, TGF-a and TGF-b could be
detected in wound fluid; monocytes and macrophages produce 10–20 times more IL-1b than IL-1a
[45–49].
The literature data mainly refer to measurements
in body fluids (serum, plasma, wound fluid, cell
culture supernatants). They clearly show up-regulation of the blood levels of various factors, especially
of IL-6, following inflammatory or traumatic stimulation [21–34]. The temporal occurrence of the
various mediators is almost invariably protracted
compared to our results (dimension of hours).
It is revealing that also in the clinical literature a
close relation between the severity of trauma or
disease course and the cytokine concentrations is
stated. Similar to our study the most significant
increases were described for the stimulator of the
acute phase reaction, IL-6 [30–32]. IL-6 can be
regarded as a key mediator for trauma, injuries and
operations [33,50–53]. Systemic levels follow locally elevated levels in wound fluids [32].
In sepsis, excessively elevated IL-6 levels were

observed persisting partially for days [6–9,28,54–
57]. On the other hand, a rapid down-regulation after
stimulation is also well-known: in experimental
animals in endotoxic shock TNF-a reached a maximum after 90 min and fell back to basal levels after
210 min [26,27].
Generally, it must be considered that plenty of
natural diseases may influence the (mainly systemic)
levels of proinflammatory cytokines (see reviews in
Refs. [11,13,58]). The local determination of the
mediators in the skin wound and, in particular, the
relation of the results to normal skin of the same
individual prevents theoretically possible falsifications.

Further influencing factors for systemic cytokine
levels are as follows: genetic disposition [59], biological age [60,61], psychological stress [62] and
pregnancy [63].
Higher age and psychological stress are able to
reduce the cytokine activation and production. In our
study we could not find a statistical dependence on
age, but some observations in single cases point into
this direction. In any case, it is evident that numerous
factors influence the complex cytokine network and
explain the high variation. However, the chosen
methodological procedure and the type of evaluation
with the formation of quotients between wound
samples and control skin avoids some of the possible
pitfalls and provides a good basis for a usable
cytokine quantitation.
Considering the prerequisites mentioned, the quantitative analysis of proinflammatory cytokines in
wound extracts can contribute to the determination of

vitality and wound age, in particular in the very early
post-traumatic interval (classic stab wounds, vitality),
but also in the later phases of the wound healing
process.

Acknowledgements
The experimental work presented in this paper was
performed at the Institute of Forensic Medicine of
the University of Cologne, Cologne, Germany. It
was supported by grants from the ‘‘Maria-Pesch¨
Stiftung’’ and the ‘‘Verein der Freunde und Forderer
¨
¨
der Universitat zu Koln’’. The authors are very
grateful to Prof. Dr. M. Paulsson, director of the
Institute of Biochemistry II, University of Cologne,
for inestimable support in the development of biochemical techniques and advice in the selection of
parameters for a useful evaluation.

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healing: Biochemical properties of growth factors and their
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