Резюме
Войченко В.В.¹, Зарицкий Г.А., Петрошак А.Ю, Гурина О.А. Возможности иммуногистохимических методов исследования для установления прижизненности травмирования кожи и мышц.
В статье проведен анализ литературных данных в отношении возможностей применения иммуногистохимических методов исследования для определения прижизненности травмы, образовавшейся в сроки ближайшие к наступлению смерти. Обращено внимание на необходимость комплексного исследовния нескольких иммуногистохимических маркеров с целью повышения достоверности получаемого результата. Рекомендовано проведение дополнительных исследований в отношении каждого из маркеров, во избежание получения ложных результатов при проведении судебно-медицинских экспертиз.
Ключевые слова: судебно-медицинская экспертиза, иммуногистохимеческие методы, травма, труп, прижизненность.
Резюме
Войченко В.В., Зарицкий Г.А., Петрошак О.Ю, Гурина О.О. Можливості імуногістохімічних методів дослідження для встановлення зажиттєвості травмування шкіри й м'язів.
У статті проведено аналіз літературних даних відносно можливостей застосування імуногістохімічних методів дослідження для визначення зажиттєвості травми, що утворилася в терміни найближчі до настання смерті. Звернено увагу на необхідність комплексного дослідження декількох імуногістохімічних маркерів з метою підвищення вірогідності результату, який отримано. Рекомендоване проведення додаткових досліджень у відношенні кожного з маркерів, щоб уникнути одержання хибних результатів при проведенні судово-медичних експертиз.
Ключові слова:судово-медична експертиза, імуногістохімічні методи, травма, труп, зажиттєвість.
Summary
Vojchenko V. V., Zaritsky G.A., Petroshak А.Y., Gurina O. A. Possibilities immunohistochemical methods of research for an establishment intravital formation of injury skin and muscles.
In article the analysis of the literary data concerning application possibilities immunohistochemical methods of research for definition of vitality a trauma formed in terms proximate to approach of death is carried out. The attention to necessity complex research several of immunohistochemical markers for the purpose of increase of reliability of received result is paid. Carrying out of additional researches concerning each of markers, in order to avoid reception of false results at carrying out of forensic medical examinations is recommended.
Key words: forensic medical examination, immunohistochemical techniques, trauma, the corpse, intravital.
Рецензент: д.мед.н., проф. В.Д. Мишалов

УДК 616.74-005.4-018.6-091.8-076.1:340.6

¹ ОКУ «Бюро судебно-медицинской экспертизы» Днепропетровской областной государственной администрации

"Bureau of forensics" Dnipropetrovsk Regional State Administration

Национальная медицинская академия последипломноого образования им. П.Л. Шупика

г. Киев, Дорогожицкая, 9, 04112, Украина

Shupyk National Medical Academy of Postgraduate Education

Kyiv, 9, Dorohozhyts’ka  str. , 04112, Ukraine

kissandr@rambler.ru

Establishment of   causal relationship between approach of death and trauma existence  is  one of the key questions while carrying out a forensic medical examination of a corpse. Basic aspects  for  solution the problem  of  trauma and death   interaction are first of all the definition of an intravital injury and limitation formation. Differentiation of intravital and postmortal injuries  and establishment of traumatizing limitation has not still lost its applicability in a forensic medical examination. The physiological answer of an organism to existence of a skin and muscular wound is a well organized biological process which classically consists of three consecutive phases –  dolor, proliferation and differentiation. Participation in each of symbolically denoted phases of  various biological substances involved in the process of wound healing  taking into account their individual activity and  combination ("temporary extinctions ") gives the chance to use in a forensic medical examination the answer of an organism to a trauma for definition of intravital and limitation traumatizing. The level of development of  modern immunohistochemical researches displaying reaction of telas on a trauma assumes establishment of the markers which are capable of objectively  differentiating intravital and postmortal injuries. In it's turn the sequence of inclusion of biologically active substances while  a wound process in  combination with degree of their immunohistochemical activity allow to judge about a trauma limitation formation. Unfortunately,  the methods which today have found broad application in domestic pathological anatomy have only informative value despite their practical prospects for forensic medicine.

We will try to present in this review the examples of the most studied immunohistochemical markers of  skin and muscular injuries suitable for application in forensic medical  diagnostics of the early posttraumatic period  - the most difficult one in forensic medical traumatology of a corpse   .

1 . Fibroneсtin is a  multipurpose cell adhesion protide   found in blood and various telas. In the process  of wound healing  fibronectin can maintain adhesion and  dynamics of fibroplasts, keratinocytes and endotheliocytes. Fibronectin is the most sensitive marker for definition of an intravital trauma , emphasising  that in certain cases immunopositive reactions of fibronectin are already produced   in a few minutes after injuring[1]. Therefore as  markers which are capable to reveal the early alternative answer to a trauma, first of all A. Fieguth, H. Feldbrugge, T. Gerich, W.J. Kleemann, H.D. Troger [2] recommend to use the methods  connected with an assessment of fibronectin immunohistochemical activity. Stable colouring of immunohistochemical preparations of injured skin and muscles is already observed in 20 minutes from the moment of a trauma up to 30 days. Consecutive decrease of immunohistochemical reaction is observed  with increase of posttraumatic period duration. 

In the phase of  skin wounds dolor  during transmigration of leukocytes  is observed the interaction between leukocytes and endothelial cells  mediated by adhesion molecules (P-selectin, E-selectin, ICAM-1, VCAM-1).

2 . Immunohistochemical researches with a semi-quantitative assessment of P-selectin have showed his high selectivity as a marker of intravital  traumatizing  since 3 minutes from injury formation for the first seven hours.

3 . Positive reaction to E-selectin begins from the first hour after the trauma moment remaining for 17 days with considerable decrease of immunohistochemical activity in 12 hours after  traumatizing [3].

4 . The ICAM-1 expression (a cell adhesion molecule   - "a cluster of differentiation 54") on a cytoplasmatic interseptum  abruptly increases on a  traumatic influence as a result of stimulation by cytokins  . Strong and moderate positive immunohistochemical colouring on ICAM-1 is observed in 90 minutes after  traumatizing and no later than 3,5 days. According to J. Dressler, L. Bachmann, M. Kasper, J.G. Hauck, E. Muller [4] a weak positive immunohistochemical answer in the intact skin  connected with specific reaction of keratinocytes  and endotheliocytes  of blood vessels to ICAM-1  sometimes can be observed .

5 . Immunohistochemical activity of VCAM-1 ("a vascular cell adhesion molecule-1 ") -  a protide of the superfamily of  immunoglobulins participating in adhesion of leukocytes and endotheliocytes at a strong and moderate luminescence displays an intravital   skin and muscles injury  since 3 hours after a trauma till 3,5 days after it [5].

6 . The immunohistochemical assessment of a wound condition concerning its intravital and emergence term  by the analysis of transforming growth factors  of TGF-α and TGF-β1 testifies the possibility of differentiation of intravital injury limitation from several minutes to 6 weeks with the maximum reliability of a post-traumatic interval till 5 hours. The increase of the TGF-α level corresponds to the epidermal responsive answer for an intravital trauma since 10 minutes  after wounding  at the maximum values in time of 30-60 minutes. TGF-β1 steadily reflects the posttraumatic answer of connective tissue . Thus positive immunohistochemical indices have already been observed by S. Vieler, B. Madea [6] in  sword-cuts  in a few minutes after intravital  traumatizing with the  same  peak of the maximum reaction of 30-60minutes  as for TGF-α .

7 . Cytokines are multipurpose glycoproteins which also take active part in  the local answer to a trauma. The representatives of this family are CXC chemokine (IL) and   tumor necrosis factor  (TNF).

Immunohistochemical representation  of an CXC chemokine -1-alpha (IL-1-α) expression in the region of skin wounds has showed a positive reaction to IL-1-α in wounds from  4 hours to 24 hours. With increase  of a wound age  reaction still remains, however reliability of an immunohistochemical assessment of an expression of IL-1-α concerning an intravital trauma is very low [7].

In W.Grellner's  work. [8] ] it is noted that the positive immunohistochemical reaction to IL-1, IL-6 and TNF in the region of intravital wounds in comparison with control samples  have been observed in terms of 30-90 minutes since traumatizing.

8 . Derivatives of arachic acid (AA) -  inflammatory mediators with  chemotactic activity in a wound process are united in the group of  leukotrienes. The research of  leukotriene B-4 (LTB4) activity  has showed that LTB4 existence in the  wound region is a well-defined evidence of vitality.  L. He, J. Zhu [9] has specified that the indicant of vitality is informative for injury differentiation less than in half an hour after  traumatizing.

9 . Possibilities of application of the immunohistochemical analysis of  D-dimer level – a specific product of  fibrin breaking  is connected first of all with an assessment of dynamics of coagulative processes in the region of  traumatizing. The increase of  D-dimer level in the region of intravital sword-cuts is observed in comparison with control in the  period beginning less than from 30 minutes from the traumatizing moment. Laboratory researches have showed selectiveness of this method to the injuries formed by  sharp objects  action and lack of any statistical disparity between control samples and portions  of scratch marks and ecchymosis [10].

10 . The researches devoted to ability of cathepsin-D – an intracellular  aspartyl protease have showed the possibility of using  this marker for fixing of  traumatic answer which most probably is connected with the pepsin like action causing nonspecific activation of other telas' cathepsins. Since 5 minutes after a trauma C. Herna´ndez-Cueto, J.A. Lorente, I. Pedal et al [11] have fixed the increase of cathepsin specific activity  in the region of injury more than by 5 times in comparison with control samples. The excess influence of  internal causes on  cathepsin-D activity in a traumatizing region  attracted special attention.

Certainly not all perspective markers of intravital skin and muscular injuries are represented in this review. The role of each of the considered factors according to various authors's opinions can have a positive effect with sufficient degree of reliability. Other researchers state doubt in method opportunities based on results of own observations.

Before using at least some of the listed methods in practice of a forensic medical examination the results from references have to be confirmed by complex scientific and practical researches. According to Kondo T. [12] ] there is a risk of excess positive reaction in postmortal injuries. Not a  less risk in our opinion is connected  with the false-negative immunohistochemical reactions caused by value of a postmortal interval, a temperature exposure of a corpse, etc.

According to the majority of the authors’ opinions  the optimality of representation of sensitivity and specificity of immunohistochemical markers of intravital  skin and muscular traumatizing is based on combination of the analysis of several groups of markers with previous careful check on big statistical groups with the verified intravital and postmortal injuries.
Литература

1. Betz P. Neue Methoden zur histologischen Altersbestim­mung menschlicher Hautwunden / P. Betz. - Schmidt-Ro¨mhild, Lu¨beck, 1996. -
2. The time-dependent expression of fibronectin, MRP8, MRP14 and defensin in surgically treated human skin wounds / A. Fieguth, H. Feldbrugge, T. Gerich [et al.] // Forensic Sci. Int. – 2003. – Vol. 131. – P. 156–161.
3. Enhanced expression of selectins in human skin wounds / J. Dressler, L. Bachmann, R. Koch, E. Muller // Int. J. Legal Med. – 1999. - Vol. 112. – P. 39–44.
4. Time dependence of the expression of ICAM-1 (CD 54) in human skin wounds / J. Dressler, L. Bachmann, M. Kasper [et al.] // Int. J. Legal Med. – 1997. - Vol. 110. – P. 299–304.
5. Expression of adhesion molecules in skin wounds: diagnostic value in legal medicine / J. Dressler, L. Bachmann, P. Strejc [et al.] // Forensic Sci. Int. – 2000. - Vol. 113. – P. 173–176.
6. Vieler S. Transforming growth factors (TGF-α and TGF-β1) in the determination of vitality and wound age: immunohistochemical study on human skin wounds / S. Vieler, B. Madea. // Forensic Science International. – 2005. – Vol. 153, Is. 2. – P. 174-180.
7. Kondo T. Immunohistochemical and morphometrical study on the temporal expression of interleukin-1a (IL-1a) in human skin wounds for forensic wound age determination / T. Kondo, T. Ohshima, W. Eisenmenger // Int. J. Legal Med. – 1990. - Vol. 112. – Р. 249–252.
8. Grellner W. Time-dependent immunohistochemical detection of proinflammatory cytokines (IL-1b, IL-6, TNF-a) in human skin wounds / W. Grellner // Forensic Sci. Int. – 2002. - Vol. 130. – Р. 90–96.
9. He L. Distinguishing antemortem from postmortem injuries by LTB4 quantification / L. He, J. Zhu // Forensic Sci. Int. – 1996. - Vol. 81. – Р. 11–16.
10.  Diagnostic ability of D-dimer in the establishment of the vitality of wounds / C. Herna´ndez-Cueto, D.N. Vieira, E. Girela [et al.] // Forensic Sci. Int. – 1995. - Vol. 76. – Р. 141–149.
11. Cathepsin D as a vitality marker in human skin wounds / C. Herna´ndez-Cueto, J.A. Lorente, I. Pedal [et al.] // Int. J. Legal Med. – 1993. - Vol. 106. – Р. 145–147.
12. Kondo T. Timing of skin wounds / T. Kondo // Legal Medicine. – 2007. - Vol. 9. – Р. 109–114.