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Expression of fibronectin suicidal in gunshot wounds
Forensic Science International 147S (2005) S5–S7 www.elsevier.com/locate/forsciint
Expression of fibronectin suicidal in gunshot wounds J. Balazˇic, A. Grajn*, E. Kralj, A. Sˇerko, B. Sˇtefanicˇ Institute of Forensic Medicine, Medical Faculty, University of Ljubljana, Korytkova 2, 1000 Ljubljana, Slovenia Available online 30 October 2004
Abstract Fibronectin has been proposed as the reliable marker of wound vitality and potent tool for age estimation in cases where survival time is at least few minutes. Cases of suicidal gunshot wounds were divided into two groups according to projectile energy and damage of the vital structures. Immunohistochemical reaction on fibronectin was evaluated with semiquantitative scale, which included sum of intensity and widespread of the reaction. Mann–Whitney test has shown statistical significant difference between groups (P 0.001). # 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Fibronectin; Wound vitality; Gunshot; Suicide
1. Introduction Question of whether the wound was sustained when the victim was still alive or already dead is still not completely answered. Determination of wound vitality and age in forensic pathology resides on the process of wound healing. This information is obtained with histopathological examination of the wound samples [1]. Information obtained from routine histological samples is rough estimation and therefore has limited forensic value. The development of immunohistochemical techniques has significantly contributed to the accuracy of the estimation. However, in daily forensic routine, the majority of sustained wound cases have relative short survival time (e.g. few seconds to minutes). In such time interval, two immunohistochemical markers are believed to be indicative for vitality and wound age estimation. Those are fibronectin and p-selectin [2]. * Corresponding author. Tel.: +386 1 524 39 03; fax: +386 1 524 38 64. E-mail address: [email protected] (A. Grajn).
Studies of fibronectin expression in wound samples on human material have shown that fibronectin is a useful vitality and age marker in all cases that have survival time more than few minutes. Report about earliest reactions range from 10 to 20 min after infliction [2]. Those findings were also supported by studies on animal models [3]. In the cases where wounds were sustained immediately before death, the pattern of staining resembled the pattern that was common in post-mortem specimens [2,4,5]. Suicidal gunshot wounds are suitable material for studies of vital reaction detection on human material in relatively short survival time. This is because these wounds are nearly standardised; in the majority of cases there is one single shot and cranio-cerebral trajectory. Survival time in cranio-cerebral gunshot wounds depends on the extent of the tissue destruction. Tissue destruction depends on energy of the projectile and elastic properties of the tissue. Brain is relatively inelastic plus it is sealed in the tight compartment of the skull vault. Thus, we assumed that in the cases of low energy projectiles (e.g. pistol ammunition), the survival time would be longer than in cases where more powerful projectile has been applied (e.g. rifles).
0379-0738/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2004.09.081
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J. Balazˇic et al. / Forensic Science International 147S (2005) S5–S7
However, death in cases of low energy projectile can be instantaneous, if the vital structures of the brain stem are damaged [6,7].
2. Materials and methods Samples of gunshot wounds were taken at autopsy at Institute of Forensic Medicine in 2001–2003. On an average, 22 h have passed from death to autopsy; bodies were kept in refrigeration unit at 4 8C. Tissue blocks were deparafinized by xylol, then rinsed in 100% alcohol and water. Procedure for fibronectin included: inhibition of endogenous peroxidaze, incubation with proteinaze to uncover the antigens and application of primary antibody (polyclonal anti rabbit fibronectin Dako Hamburg, dilution 1:400). For evaluation of the fibronectin reaction, we devised a semi quantitative scale, which measures intensity and widespread of the reaction. Final scores were obtained with subtraction of extent and intensity sums from wound and opposite side of specimens. Cases were sorted into two groups. In first group, we placed cases in which we assume extremely short survival times either due to the powerful projectiles (rifles, hunting carbines or certain more powerful pistol ammunitions e.g. TT org magnums) or due to the intracranial track by where the vital structures of brain stem were damaged. Some cases from this group share both characteristics. One slide from each group was stained without antibody to serve as negative control. In second group, we placed the remaining cases. Difference in scores was tested with Mann–Whitney test (Table 1).
3. Results The cases with shorter and longer survival time are represented in Tables 2 and 3.
Table 2 Cases with shorter survival time
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Intracranial missile track
Scores
Occipito-frontal Transtemporal Transtemporal Transtemporal Baseo-parietal Baseo-parietal Transtemporal Fronto-occipital Baseo-parietal Baseo-occipital Baseo-occipital Baseo-occipital Transtemporal Baseo-occipital Baseo-occipital Transtemporal Baseo-occipital Baseo-occipital Baseo-occipital Baseo-occipital
0 2 1 1 0 0 0 0 2 0 1 1 0 0 0 0 1 0 0 0
Table 3 Cases with longer survival time
1 2 3 4 5 6 7 8 9 10
Intracranial missle track
Scores
Trans-temporal Trans-temporal Trans-temporal Trans-temporal Trans-temporal Trans-temporal Trans-temporal Trans-temporal Occipito-frontal Extra-cerebral
4 5 4 5 3 4 3 5 2 4
Mann–Whitney test has shown statistically significant difference between groups (P 0.001).
4. Discussion Table 1 Semi quantitative scores for evaluating fibronectin reaction Intensity of reaction
Points
No reaction Mild Moderate Intense
0 1 2 3
Widespread of reaction Margins only Up to 1/4 of 100 visual field Up to 1/2 of 100 visual field More than 1/2 of 100 visual field
0 1 2 3
The gunshot wounds in the head have, in general, short survival time; however, in certain conditions, the survival time is long enough for fibronectin reaction to develop. The cases with high scores (Table 3) share the following characteristics: projectile has relatively low energy, and the vital structures in brain stem were spared from destruction. Survival time must have been long enough for fibronectin reaction to occur. Infiltration with polymorphonuclear granulocytes was minor and presented only in one case. Intracerebral missile track in most cases from Table 3 was transtemporal. In this way, vital structures of the brain stem were in the shadow of anterior fossa and region of the sella
J. Balazˇic et al. / Forensic Science International 147S (2005) S5–S7
turcica, where significant amount of energy has been absorbed [6,7]. The variation between scores is due to the simple fact that this type of study cannot be carried out in controlled laboratory conditions. Ammunition manufacturers were different so as the storage conditions. Furthermore, missile track was not standard; position of entrance wound and angle of firing were variable among cases. In cases where extremely short survival or instantaneous death was assumed (Table 3), the unambiguous reaction was absent; however, in few we observed pattern that was described in post-mortem specimens and also in survival times of few seconds. It included week staining at the margins of the wounds [4,5]. Staining on the edges can also be attributed to drying; thus, we evaluated reaction on both sides of the specimen with subsequent subtraction of scores. Areas of bleeding were excluded from evaluation. In only 1 case from each group soft point projectile was used. In all remaining were standard full metal jacket bullets. Autolysis can cause false negative and false positive reactions [8]. Most bodies were discovered shortly after death. Signs of putrefaction were absent in all cases. Thus, the effect of autolysis is negligible.
5. Conclusions Results support indications from earlier studies that fibronectin is reliable marker of wound vitality and age in wounds that have relative short survival time (e.g. few
S7
minutes) before the evident polymorphonuclear infiltration occurs [3–5]. In extremely shot survival time, the reaction is either not present, or it cannot be unambiguously distinguished from post-mortem reaction patterns.
References [1] T. Oshima, Forensic wound examination, Forensic Sci. Int. 113 (2000) 153–164. ¨ bersichtsreferat: histologische kriterien zur alter[2] P. Betz, U sscha¨ tzung menschlicher hautwunden, Z. Rechtsmedizin 9 (1999) 163–169. [3] W. Grellner, S. Dimmeler, B. Madea, Immunohistochemical detection of fibronectin in postmortem incised wounds of porcine skin, Forensic Sci. Int. 97 (2–3) (1998) 109–116. [4] P. Betz, A. Nerlich, J. Wilske, et al. Immunohistochemical localization of fibronectin as a tool for the age determination of human skin wounds, Int. J. Legal Med. 105 (1992) 21–26. [5] P. Betz, A. Nerlich, J. Wilske, et al. The immunohistochemical localization of alpha-1-antichymotripsin and fibronectin and its meaning for the determination of the vitality of human skin wounds, Int. J. Legal Med. 105 (1993) 223–227. [6] B. Karger, Penetrating gunshots to the head and lack of immediate incapacitation. I. Wound ballistics and mechanisms of incapacitation, Int. J. Legal Med. 108 (1995) 53–61. [7] B. Karger, Penetrating gunshots to the head and lack of immediate incapacitation. II. Review of case reports, Int. J. Legal Med. 108 (1995) 117–126. [8] A. Fieguth, W.J. Kleemann, von Wasielevski, et al. Influence of postmortem changes immunohistochemical reactions in skin, Int. J. Legal Med. 110 (1997) 18–21.
Expression of fibronectin suicidal in gunshot wounds J. Balazˇic, A. Grajn*, E. Kralj, A. Sˇerko, B. Sˇtefanicˇ Institute of Forensic Medicine, Medical Faculty, University of Ljubljana, Korytkova 2, 1000 Ljubljana, Slovenia Available online 30 October 2004
Abstract Fibronectin has been proposed as the reliable marker of wound vitality and potent tool for age estimation in cases where survival time is at least few minutes. Cases of suicidal gunshot wounds were divided into two groups according to projectile energy and damage of the vital structures. Immunohistochemical reaction on fibronectin was evaluated with semiquantitative scale, which included sum of intensity and widespread of the reaction. Mann–Whitney test has shown statistical significant difference between groups (P 0.001). # 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Fibronectin; Wound vitality; Gunshot; Suicide
1. Introduction Question of whether the wound was sustained when the victim was still alive or already dead is still not completely answered. Determination of wound vitality and age in forensic pathology resides on the process of wound healing. This information is obtained with histopathological examination of the wound samples [1]. Information obtained from routine histological samples is rough estimation and therefore has limited forensic value. The development of immunohistochemical techniques has significantly contributed to the accuracy of the estimation. However, in daily forensic routine, the majority of sustained wound cases have relative short survival time (e.g. few seconds to minutes). In such time interval, two immunohistochemical markers are believed to be indicative for vitality and wound age estimation. Those are fibronectin and p-selectin [2]. * Corresponding author. Tel.: +386 1 524 39 03; fax: +386 1 524 38 64. E-mail address: [email protected] (A. Grajn).
Studies of fibronectin expression in wound samples on human material have shown that fibronectin is a useful vitality and age marker in all cases that have survival time more than few minutes. Report about earliest reactions range from 10 to 20 min after infliction [2]. Those findings were also supported by studies on animal models [3]. In the cases where wounds were sustained immediately before death, the pattern of staining resembled the pattern that was common in post-mortem specimens [2,4,5]. Suicidal gunshot wounds are suitable material for studies of vital reaction detection on human material in relatively short survival time. This is because these wounds are nearly standardised; in the majority of cases there is one single shot and cranio-cerebral trajectory. Survival time in cranio-cerebral gunshot wounds depends on the extent of the tissue destruction. Tissue destruction depends on energy of the projectile and elastic properties of the tissue. Brain is relatively inelastic plus it is sealed in the tight compartment of the skull vault. Thus, we assumed that in the cases of low energy projectiles (e.g. pistol ammunition), the survival time would be longer than in cases where more powerful projectile has been applied (e.g. rifles).
0379-0738/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2004.09.081
S6
J. Balazˇic et al. / Forensic Science International 147S (2005) S5–S7
However, death in cases of low energy projectile can be instantaneous, if the vital structures of the brain stem are damaged [6,7].
2. Materials and methods Samples of gunshot wounds were taken at autopsy at Institute of Forensic Medicine in 2001–2003. On an average, 22 h have passed from death to autopsy; bodies were kept in refrigeration unit at 4 8C. Tissue blocks were deparafinized by xylol, then rinsed in 100% alcohol and water. Procedure for fibronectin included: inhibition of endogenous peroxidaze, incubation with proteinaze to uncover the antigens and application of primary antibody (polyclonal anti rabbit fibronectin Dako Hamburg, dilution 1:400). For evaluation of the fibronectin reaction, we devised a semi quantitative scale, which measures intensity and widespread of the reaction. Final scores were obtained with subtraction of extent and intensity sums from wound and opposite side of specimens. Cases were sorted into two groups. In first group, we placed cases in which we assume extremely short survival times either due to the powerful projectiles (rifles, hunting carbines or certain more powerful pistol ammunitions e.g. TT org magnums) or due to the intracranial track by where the vital structures of brain stem were damaged. Some cases from this group share both characteristics. One slide from each group was stained without antibody to serve as negative control. In second group, we placed the remaining cases. Difference in scores was tested with Mann–Whitney test (Table 1).
3. Results The cases with shorter and longer survival time are represented in Tables 2 and 3.
Table 2 Cases with shorter survival time
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Intracranial missile track
Scores
Occipito-frontal Transtemporal Transtemporal Transtemporal Baseo-parietal Baseo-parietal Transtemporal Fronto-occipital Baseo-parietal Baseo-occipital Baseo-occipital Baseo-occipital Transtemporal Baseo-occipital Baseo-occipital Transtemporal Baseo-occipital Baseo-occipital Baseo-occipital Baseo-occipital
0 2 1 1 0 0 0 0 2 0 1 1 0 0 0 0 1 0 0 0
Table 3 Cases with longer survival time
1 2 3 4 5 6 7 8 9 10
Intracranial missle track
Scores
Trans-temporal Trans-temporal Trans-temporal Trans-temporal Trans-temporal Trans-temporal Trans-temporal Trans-temporal Occipito-frontal Extra-cerebral
4 5 4 5 3 4 3 5 2 4
Mann–Whitney test has shown statistically significant difference between groups (P 0.001).
4. Discussion Table 1 Semi quantitative scores for evaluating fibronectin reaction Intensity of reaction
Points
No reaction Mild Moderate Intense
0 1 2 3
Widespread of reaction Margins only Up to 1/4 of 100 visual field Up to 1/2 of 100 visual field More than 1/2 of 100 visual field
0 1 2 3
The gunshot wounds in the head have, in general, short survival time; however, in certain conditions, the survival time is long enough for fibronectin reaction to develop. The cases with high scores (Table 3) share the following characteristics: projectile has relatively low energy, and the vital structures in brain stem were spared from destruction. Survival time must have been long enough for fibronectin reaction to occur. Infiltration with polymorphonuclear granulocytes was minor and presented only in one case. Intracerebral missile track in most cases from Table 3 was transtemporal. In this way, vital structures of the brain stem were in the shadow of anterior fossa and region of the sella
J. Balazˇic et al. / Forensic Science International 147S (2005) S5–S7
turcica, where significant amount of energy has been absorbed [6,7]. The variation between scores is due to the simple fact that this type of study cannot be carried out in controlled laboratory conditions. Ammunition manufacturers were different so as the storage conditions. Furthermore, missile track was not standard; position of entrance wound and angle of firing were variable among cases. In cases where extremely short survival or instantaneous death was assumed (Table 3), the unambiguous reaction was absent; however, in few we observed pattern that was described in post-mortem specimens and also in survival times of few seconds. It included week staining at the margins of the wounds [4,5]. Staining on the edges can also be attributed to drying; thus, we evaluated reaction on both sides of the specimen with subsequent subtraction of scores. Areas of bleeding were excluded from evaluation. In only 1 case from each group soft point projectile was used. In all remaining were standard full metal jacket bullets. Autolysis can cause false negative and false positive reactions [8]. Most bodies were discovered shortly after death. Signs of putrefaction were absent in all cases. Thus, the effect of autolysis is negligible.
5. Conclusions Results support indications from earlier studies that fibronectin is reliable marker of wound vitality and age in wounds that have relative short survival time (e.g. few
S7
minutes) before the evident polymorphonuclear infiltration occurs [3–5]. In extremely shot survival time, the reaction is either not present, or it cannot be unambiguously distinguished from post-mortem reaction patterns.
References [1] T. Oshima, Forensic wound examination, Forensic Sci. Int. 113 (2000) 153–164. ¨ bersichtsreferat: histologische kriterien zur alter[2] P. Betz, U sscha¨ tzung menschlicher hautwunden, Z. Rechtsmedizin 9 (1999) 163–169. [3] W. Grellner, S. Dimmeler, B. Madea, Immunohistochemical detection of fibronectin in postmortem incised wounds of porcine skin, Forensic Sci. Int. 97 (2–3) (1998) 109–116. [4] P. Betz, A. Nerlich, J. Wilske, et al. Immunohistochemical localization of fibronectin as a tool for the age determination of human skin wounds, Int. J. Legal Med. 105 (1992) 21–26. [5] P. Betz, A. Nerlich, J. Wilske, et al. The immunohistochemical localization of alpha-1-antichymotripsin and fibronectin and its meaning for the determination of the vitality of human skin wounds, Int. J. Legal Med. 105 (1993) 223–227. [6] B. Karger, Penetrating gunshots to the head and lack of immediate incapacitation. I. Wound ballistics and mechanisms of incapacitation, Int. J. Legal Med. 108 (1995) 53–61. [7] B. Karger, Penetrating gunshots to the head and lack of immediate incapacitation. II. Review of case reports, Int. J. Legal Med. 108 (1995) 117–126. [8] A. Fieguth, W.J. Kleemann, von Wasielevski, et al. Influence of postmortem changes immunohistochemical reactions in skin, Int. J. Legal Med. 110 (1997) 18–21.