Histological analysis of short-term vital reactions in skin wounds:

potential applications in forensic work

Obac, AR.a*, Carvalho, EG.b, Silva, PCS.c, Fenerich-Verani, N.d and Almeida, M.e
Universidade Federal de São Paulo – UNIFESP, Rua Borges Lagoa, 908, Vila Clementino, São Paulo, SP, Brazil
a

b
Departamento de Ecologia e Biologia Evolutiva, Universidade Federal de São Carlos – UFSCar,
Rod. Washington Luís, Km 235, CP 676, CEP 13605-905, São Carlos, SP, Brazil
c
Departamento de Patologia, Universidade Federal de São Paulo – UNIFESP,
Rua Botucatu, 740, Vila Clementino, CEP 04023-900, São Paulo, SP, Brazil
d
Departamento de Hidrobiologia, Universidade Federal de São Carlos – UFSCar,
Rod. Washington Luís, Km 235, CP 676, CEP 13605-905, São Carlos, SP, Brazil
e
Departamento de Medicina Legal, Universidade Federal de São Paulo – UNIFESP,
Rua Botucatu, 740, Vila Clementino, CEP 04023-900, São Paulo, SP, Brazil
*e-mail: alexobac@hotmail.com
Received July 20, 2010 – Accepted October 1, 2010 – Distributed November 30, 2011
(With 1 figure)

Abstract
In forensic medical work, in cases with homicide suspicion, it is important to be able to determine with the greatest
possible precision when injuries occurred and whether during vital, or post-mortem conditions. Although several
markers of vitality can be employed, it has been attested that components of the extra-cellular matrix, such as fibrin,
are among the earliest to be evidenced. In the present study, the histological-histochemical Mallory’s Trichrome
staining method, previously selected was tested to determine the presence of vital reaction in Wistar rats through fibrin
accumulation by testing three short reaction time intervals: 15, 30 and 60 minutes after the skin wound infliction. For
all time intervals tested, including the shortest (15 minutes), the presence of fibrin at the edges of the skin wound was
evidenced. The accumulation of fibrin was, nevertheless, more pronounced at 30 and 60 minutes after the wound. It
could be concluded that fibrin is a good marker for vital reaction and that it can be detected very early, within a few
minutes after the injury. It is proposed that histological method coupled to the histochemical staining technique here
tested can be incorporated into routine forensic work as a tool for evidencing the existence or not of vital reaction.
Keywords: vital reaction, skin wound, histological analysis, fibrin.

Análise histológica da reação vital em ferimentos da pele em curto intervalo de tempo:

aplicações potenciais em trabalho forense

Resumo
No trabalho médico-forense, um aspecto importante em casos de suspeita de homicídios é a determinação, com a
maior precisão possível, de quando ocorreram os ferimentos e se em condições de plena vitalidade ou se post-mortem.
Embora diversos marcadores possam ser utilizados para o diagnóstico de vitalidade das lesões, alguns componentes da
matriz extracelular, como a fibrina, podem ser os primeiros sinais de reação vital a serem evidenciados. No presente
estudo, o método de coloração histológica-histoquímica Tricrômio de Mallory, previamente selecionado, foi testado
para determinar a presença de reação vital em ratos Whistar por meio do acúmulo da fibrina, testando-se três tempos
curtos de reação: 15 , 30 e 60 minutos após a realização de ferimento na pele. Para todos os tempos testados, inclusive
no tempo mais curto (15 minutos) foi evidenciado o acúmulo da fibrina na região próxima à borda do ferimento. O
acúmulo de fibrina foi, no entanto, mais intenso 30 e 60 minutos após a ocorrência da lesão. Pôde-se concluir que a
fibrina é um bom marcador para a reação vital, podendo ser detectada muito cedo, poucos minutos após a ocorrência do
ferimento. Propõe-se que a técnica histológica acoplada à técnica histoquímica da coloração pelo Tricrômio de Mallory
poderia ser facilmente incorporada à rotina do trabalho médico forense, como ferramenta para evidenciar a existência
ou não de reação vital.
Palavras-chave: reação vital, ferimento da pele, análise histológica, fibrina.

Braz. J. Biol., 2011, vol. 71, no. 4, p. 1011-1014 1011

 Obac, AR. et al.

1. Introduction analysis or because they are very expensive. Histological

and histochemical techniques could be a better approach
When a wound is inflicted on a living organism, a considering its simplicity and relatively low cost.
series of events is triggered, called vital reaction. Vital In the present study, Mallory´s trichrome staining
reaction is a complex mechanism, involving a number of technique was tested regarding its potential to show the
phenomena occurring over distinct spatial and temporal vital reaction following skin injuries and the short-term
scales. Determination of wound vitality and the interpretation chronology of the wounds using fibrin as a marker of
of the chronological sequence of events is an important vital reaction.
part of the work in forensic medicine. It is important to
be able to report with the greatest possible precision when
injuries occurred and whether during the vital, or during 2. Materials and Methods
the postmortem period (Ohshima, 2000; Kondo, 2007). An experiment to compare the vital reaction
A characteristic of living tissue is its capacity to respond intensity was performed at three different time intervals:
to external stimuli. When the stimulus is a traumatic 15, 30 and 60 minutes after injury and non-injured (control).
aggression whether physical, chemical or biological, the Twelve Wistar rats weighing 200-300 g. were used
tissue reaction consists essentially of an acute inflammation, (3 replicates in each treatment). They were kept in cages,
usually proportional to the magnitude of the trauma in groups of three for 24 hours before the experiment with
(Cardillo et al., 2005). free access to food and water. The rats were anesthetised
Recently it has been shown that since certain reactions by intramuscular injection of a mixture of ketamine
do not occur in postmortem wounds, while in life there hydrochloride and xylazine hydrochloride in the proportion
is a complex and organized sequence of events linked of 3:2 in a dosage of 0.1 mL/100 g body weight.
to tissue repair and restoration of the function of injured Hair was removed from the rat bellies with a razor
organs, making it possible to characterize, step by step, and the area washed with physiological solution and dried
morphological and functional stages, and enabling the with sterile cloth. Each animal was anesthetised and a
creation of a model that could be applied to many problems 0.5 cm long incision was made in the prepared area of
in legal or forensic medicine (Oehmichen and Kirchner skin with a scalpel. After 15 minutes, a 1 cm2 skin sample
1996; Ortiz-Rey et al., 2002, 2003; Oehmichen, 2004; was removed from the animal in the first group, with the
Kondo, 2007). incision at the center. The material was preserved in 10%
The determination of wound age or wound vitality is very formalin. This procedure was then repeated after 30 minutes
important in forensic practice. Analysis of wound vitality for the second group and after 60 minutes for the third.
markers, such as the inflammatory reaction mediators, are The control group was then anesthetised and sacrificed to
essential for diagnosing the vitality of the lesions and to obtain a post-mortem sample.
establish lesion chronology, pre and post-mortem. Although The material was subjected to histological analysis,
a classical approach, this is still an area of concern in modern being fixed, stained and sectioned according to standard
forensic medicine and there is a continuous demand for routine methods in the Histology laboratory of the
further research and gathering of information to be applied Department of Hydrobiology, Federal University of São
in daily practical work (Grellner and Burkhard, 2007).
Carlos (UFSCar). The tissue was dehydrated through
Earlier studies carried out by Fatteh (1966, 1971)
xylene and hydrated through ethanol series and transferred
showed that morphological parameters detectable by
to xylene (3 × 15 minutes) and subsequently embedded in
routine histological methods could be used to estimate
melted paraffin at 58 °C, for 24 hours at room temperature.
wound age in human skin, with valuable applications in
Skin samples were oriented parallel to the incision, allowed
legal medicine.
to harden in paraffin for two days and then cut into 8 mm
Various molecules can act as markers of vitality.
sections on a rotary microtome. Serial sections from each
Proteins of the extracellular matrix (fibrinogen, fibrin,
fibronectin, tenascin and vitronectin) can be used as specimen were obtained by microtomy and stained by the
markers of vitality at time intervals varying from a few Mallory’s Trichrome method (Bancroft and Stevens, 1996)
minutes up to 10 hours after the lesion (Oehmichen, 2004). Serial sections were qualitatively evaluated for the presence
The structural proteins are among the molecules detected of fibrin, using a computer image acquisition system (Carl
earliest at the edge of the wound, in the post-traumatic Zeiss microscope Axioscop 2 plus Axiocam camera). The
period and recent work indicates that fibrin can be detected undamaged margin beside the incision in the control group
in a time interval as short as 5 minutes after the wound is was used as control and the injured tissue, left to react for
inflicted (Kondo, 2007). various times (15 , 30 and 60 minutes after injury) was
Fibrin is an important element of the exudate of acute evaluated for this vital reaction marker.
inflammatory responses and can be found wherever there is
recent tissue damage. In the injuries performed after fatal 3. Results
trauma, fibrin is very weakly detected being found in trace
amounts or artifacts at edge of the wound (Oehmichen Selected skin section images for the reaction time of
and Kirchner, 1996). 30 minutes and for the post-mortem incision are displayed
Although several techniques are known to be capable (Figure 1a,b). In the section representing the 30 minutes
of evidencing vital reaction they are of difficult application reaction time the fibrin accumulation at the borders of the
in the routine legal medical work due to the complexity of incision appears stained in red by the Mallory’s Trichrome

1012 Braz. J. Biol., 2011, vol. 71, no. 4, p. 1011-1014

 Short-term vital reactions in skin wounds

a b

c d

Figure 1. Fibrin accumulation at various vital reaction time intervals as red stained by Mallory’s trichrome histochemical
method: a) in vivo 30 minutes vital reaction; b) post-mortem (after 30 minutes time interval) skin section stained by Mal-
lory’s trichrome; c) in vivo 15 minutes vital reaction; d) in vivo 60 minutes vital reaction . Arrows show the accumulation
of fibrin in the wound edge.

stain mixture (Figure 1a); On the other hand, there is no 4. Discussion

fibrin accumulation in the borders of the incision inflicted
after death (Figure 1b) and the whole tissue appears It has been demonstrated that skin-wound healing
stained in blue. starts immediately after the injury and consists of a
In the sections taken from rats exposed to 15 minutes vital well-orchestrated sequence of events with three phases:
reaction, it is already possible to observe the accumulation inflammation, proliferation and maturation. In our preliminary
of fibrin at the wound edges (Figure 1c), coloured in red tests it was possible to observe that all histochemical and
by Mallory´s trichome. In the 60 minutes reaction time histological staining methods used were more efficient at
(Figure 1d), the skin section has a greater accumulation of showing presence of fibrin than of mucopolyssacharydes.
fibrin at the borders of the incision which is more evident Fibrin is an insoluble fibrous protein formed by the
than in the 15 minutes reaction time as indicated by a polymerisation of fibrinogen, a smaller soluble blood
stronger stained and extended area. protein. Fibrin is most commonly seen in recently damaged

Braz. J. Biol., 2011, vol. 71, no. 4, p. 1011-1014 1013

 Obac, AR. et al.

tissues and in acute inflammatory reaction, resulting from BRADBURY, P. and ERA, K., 1996. Connective Tissues and
oozing of fluid and plasma proteins out of damaged skin Stains. In: BANCROFT, JD. and STEVENS, A. (Eds.). Theory
vessels (Bradbury and Era, 1996). There is an overlap in and Practice of Histological Techniques. 4nd ed. Edinburgh:
Churchill Livingstone. 766 p. chap. 7, p. 113-138.
the early phase of inflammation following trauma and
alterations within the first 30 minutes can be regarded as CARDILLO, GZ., GIMENE, MP. and CAMARGO, RS., 2005.
either vital reactions or signs of wound healing (Oehmichen Marcadores inflamatórios aplicados à Medicina. News Lab,
and Kirchner, 1996). vol. 71, p. 116-128.
In paraffin sections, fibrin is strongly eosinophilic FATTEH, A., 1966. Histochemical distinction between antemortem
(Bancroft and Stevens, 1996; Bancroft and Gamble, and postmortem skin wounds. Journal of Forensic Sciences,
2005). In this study, the edge of the wound appeared red in vol. 11, no. 1, p. 17-26.
Mallory´s trichrome staining showing that fibrin deposition
-, 1971. Distinction between antemortem and mortem wounds: a
was characteristic of the initial period after the lesion. study of elastic fiber in human skin. Journal of Forensic Sciences,
A gradual and progressive staining of fibrin due to the vol. 16, no. 3, p. 393-396.
vital reaction was observed, which depended on the reaction
GRELLNER, W. and BURKHARD, M.,  2007. Demands on
time. The greatest differences were found when comparing
scientific studies: vitality of wounds and wound age estimation.
sections from 15 with those with 30 or 60 minutes of
Forensic Science International, vol. 165, no. 2-3, p. 150-154.
reaction. The vital reaction was visible in all sections after PMid:16806766. http://dx.doi.org/10.1016/j.forsciint.2006.05.029
the 15 minutes period of reaction, but not in any of those
from the post-mortem incision. Vital reaction was present KONDO, T., 2007. Timing of skin wounds. Legal Medicine, vol. 9,
no. 2, p. 109-114. PMid:17275383. http://dx.doi.org/10.1016/j.
at the edge of the skin wound, and appeared in all three
legalmed.2006.11.009
reaction times, indicating that it is feasible to distinguish
between lesions performed in vivo from those performed OEHMICHEN, M., 2004. Vitality and time course of wounds.
after death even in such short time of vital reactions. Forensic Science International, vol. 144, no. 2- 3, p. 221-31.
The occurrence of vital reaction was evident in all tests OEHMICHEN, M. and KIRCHNER, H., 1996. The Wound Healing
performed. Fibrin was a good marker of vital reaction being Process - Forensic Pathological Aspects. Lübeck: Schmidt-Römhild.
present in the skin wounds just 15 minutes after the injury.
OHSHIMA, T., 2000. Forensic wound examination. Forensic
Most studies in the literature suggested that histochemical
Science International, vol. 113, no. 1-3, p. 153-164. http://dx.doi.
markers of vital reaction will only appear some hours after org/10.1016/S0379-0738(00)00269-3
the injury. In this study we found evidence that a vitality
marker such as fibrin can be detected much earlier, within ORTIZ-REY, JA., SUÁREZ-PEÑARANDA, JM., DA SILVA,
a few minutes of the injury, and easily introduced into EA., MUÑOZ, JI., SAN MIGUEL-FRAILE, P., DE LA
FUENTE-BUCETA, A. and CONCHIERO-CARROL, L., 2002
forensic work.
Immunohistochemical detection of fibronectin and tenascin in
incised human skin injuries. Forensic Science International, vol. 126,
References p. 118-122. http://dx.doi.org/10.1016/S0379-0738(02)00032-4
ORTIZ-REY, JA., SUÁREZ-PEÑARANDA, JM., MUÑOZ-
BANCROFT, JD. and GAMBLE, M. (Eds.)., 2005. Theory and
BARÚS, JI., ÁLVAREZ, C., SAN MIGUEL, P., RODRÍGUEZ-
Practice of Histological Techniques. 5nd ed. Edinburgh: Churchill
Livingstone. 796 p. CALVO, MS. and CONCHEIRO-CARRO, L., 2003. Expression
of fibronectin and tenascin as a demonstration of vital reaction
BANCROFT, JD. and STEVENS, A. (Eds.)., 1996. Theory and in rat skin and muscle. International Journal of Legal Medicine,
Practice of Histological Techniques. 4nd ed. Edinburgh: Churchill vol. 117, no. 6, p. 356-360. PMid:14586623. http://dx.doi.
Livingstone. 766 p. org/10.1007/s00414-003-0403-6

1014 Braz. J. Biol., 2011, vol. 71, no. 4, p. 1011-1014