A NEW METHOD TO DETERMINE WOUND AGE IN HUMAN SKIN INJURIES

Authors

  • Natasha Bitoljanu Institute of Forensic Medicine, Criminalistics and Medical Deonthology, Faculty of Medicine, Ss. Cyril and Methodius University, Skopje, Republic of North Macedonia
  • Sasho Risteski Institute of Forensic Medicine, Criminalistics and Medical Deonthology, Faculty of Medicine, Ss. Cyril and Methodius University, Skopje, Republic of North Macedonia
  • Viktorija Belakaposka Srpanova Institute of Forensic Medicine, Criminalistics and Medical Deonthology, Faculty of Medicine, Ss. Cyril and Methodius University, Skopje, Republic of North Macedonia
  • Ana Ivcheva Institute of Forensic Medicine, Criminalistics and Medical Deonthology, Faculty of Medicine, Ss. Cyril and Methodius University, Skopje, Republic of North Macedonia
  • Ljupco Cakar Institute of Forensic medicine, criminalistics and medical deonthology, Faculty of Medicine, University ,,Saints Cyril and Methodius”
  • Goran Pavlovski Institute of Forensic Medicine, Criminalistics and Medical Deonthology, Faculty of Medicine, Ss. Cyril and Methodius University, Skopje, Republic of North Macedonia
  • Aleksandar Stankov Institute of Forensic Medicine, Criminalistics and Medical Deonthology, Faculty of Medicine, Ss. Cyril and Methodius University, Skopje, Republic of North Macedonia
  • Svetlana Krstevska Balkanov University Clinic for Hematology, Faculty of Medicine, Ss. Cyril and Methodius University, Skopje, Republic of North Macedonia
  • Zlatko Jakovski Institute of Forensic Medicine, Criminalistics and Medical Deonthology, Faculty of Medicine, Ss. Cyril and Methodius University, Skopje, Republic of North Macedonia

Keywords:

wound age; Real-time PCR; autopsy cases; human dermal injuries; Il-6

Abstract

Wound age determination is a significant part of everyday practice in forensic medicine. Determining the wound age answers a large number of questions arising from a criminal-legal event. The RT-PCR technique enables qualitative and quantitative analysis of mRNA and can register minimal changes in gene expression, which makes it more sensitive and accurate compared to the immunohistochemical method.

The study included 51 wound samples of autopsy cases. In this study we conducted comparative analyses for different methods of homogenization and isolation. An analysis was carried out for the selection of a suitable reference gene that will be used for normalization of the expression levels of the investigated target gene. Two reference genes, GAPDH and 18S, were analyzed in the study.

 A biphasic expression of IL-6 information RNA (mRNA IL-6) was determined in relation to the other groups, including the control group. 18S has better efficiency of amplification then GAPDH.

 RNA was isolated from all examined tissues with the TriXact kit on the MagCore Plus II instrument. It can also be concluded that mechanical maceration of the tissue was more effective than the other methods of homogenization. 18S is a more stable reference gene for the analysis of skin samples. IL-6 can could be used as a marker in wound age determination.

References

Takamiya M, Fujita S, Saigusa K, Aoki S. A study on mRNA expressions of interleukin 10 during fracture healing for wound age determination. Leg Med (Tokyo). 2008; 10(3): 131-137. doi: 10.1016/j.legalmed.2007.11.003.

Kondo T. Timing of skin wounds. Leg Med 2007; 9(2): 109-114. doi: 10.1016/j. legalmed.2006.11.009.

Grellner W, Madea B. Demands on scientific studies: Vitality of wounds and wound age estimation. Forensic Sci Int 2007; 165(2-3): 150-154. doi: 10.1016/ j.forsciint.2006.05.029.

Kondo T, Ohshima T, Eisenmenger W. Immunohistochemical and morphometrical study on the temporal expression of interleukin-1a (IL-1a) in human skin wounds for forensic wound age determination. Int J Legal Med 1999; 112(4): 249-252. doi: 10.1007/s004140050244.

Oehmichen M. Vitality and time course of wounds. Forensic Sci Int 2004; 144(2-3): 221-231. doi: 10.1016/j.forsciint.2004.04.057.

Schwartz AJ, Ricci LR. How accurately can bruises be aged in abused children? Literature review and synthesis. Pediatrics 1996; 97: 254-257.

Cecchi R. Estimationg wound age: looking into the future. Int J Legal Med 2010; 124(6): 523-536. doi: 10.1007/s00414-010-0505-x.

Bauer M. RNA in forensic science. Forensic Sci Int Genet 2007; 1(1): 69-74. doi: 10.1016/j.fsigen.2006.11.002.

Ohshima T. Forensic wound examination. Forensic Sci Int 2000; 113(1-3): 153-164. doi: 10.1016/s0379-0738(00)00269-3.

Chapman JR, Waldenström J. With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies. PLoS One 2015; 10(11): e0141853. doi: 10.1371/journal.pone.0141853.

Pool JG: Normal hemostatic mechanisms: a review. Am J Med Technol 1977; 43(8): 776-780. PMID: 888856.

Kondo T, Ishida Y. Molecular pathology of wound healing. Forensic Sci Int 2010; 203(1-3): 93-98. doi: 10.1016/j.forsciint.2010.07.004.

Enoch S, Leaper D. Basic science of wound healing. Surgery 2008; 26(2): 31-37. https://doi.org/10.1016/j.mpsur.2007.11.005.

Martin P. Wound healing--aiming for perfect skin regeneration. Science 1997; 276 (5309): 75-81. doi: 10.1126/science.276.5309.75.

Singer AJ, Clark RA. Cutaneous wound healing. N Engl J Med. 1999; 341(10): 738-746. doi: 10.1056/NEJM199909023411006.

Ma WX, Yu TS, Fan YY, Zhang ST, Ren P, Wang SB, et al. Time-dependent expression and distribution of monoacylglycerol lipase during the skin-incised wound healing in mice. Int J Legal Med 2011; 125(4): 549-558. doi: 10.1007/s00414-011-0567-4.

Derveaux S, Vandesompele J, Hellemans J. How to do successful gene expression analysis using real-time PCR. Methods 2010; 50(4): 227-230. doi: 10.1016/j.ymeth.2009.11.001.

Méndez V, Avelar E, Morales A, Cervantes M, Araiza A, González D. A rapid protocol for purification of total RNA for tissues collected from pigs at a slaughterhouse. Genet Mol Res 2011; 10(4): 3251-3255. doi: 10.4238/2011.December.22.3.

Auer H, Mobley JA, Ayers LW, Bowen J, Chuaqui RF, Johnson LA, et al. The effects of frozen tissue storage conditions on the integrity of RNA and protein. Biotech Histochem 2014; 89(7): 518-528. doi: 10.3109/10520295.2014.904927.

Mutter GL, Zahrieh D, Liu C, Neuberg D, Finkelstein D, Baker HE et al. Comparison of frozen and RNALater solid tissue storage methods for use in RNA expression microarrays. BMC Genomics 2004; 5:88. doi.org/10.1186/1471-2164-5-88

Lou JJ, Mirsadraei L, Sanchez DE, Wilson RW, Shabihkhani M, Lucey GM, et al. A review of room temperature storage of biospecimen tissue and nucleic acids for anatomic pathology laboratories and biorepositories. Clin Biochem 2014; 47(4-5): 267-273. doi: 10.1016/j.clinbiochem.2013.12.011.

Gardner H, Shearstone JR, Bandaru R, Crowell T, Lynes M, Trojanowska M, et al. Gene profiling of scleroderma skin reveals robust signatures of disease that are imperfectly reflected in the transcript profiles of explanted fibroblasts. Arthritis Rheum 2006; 54(6): 1961-1973. doi: 10.1002/art.21894.

Berglund SR, Schwietert CW, Jones AA, Stern RL, Lehmann J, Goldberg Z. Optimized methodology for sequential extraction of RNA and protein from small human skin biopsies. J Invest Dermatol. 2007 Feb;127(2):349-53. doi: 10.1038/sj.jid.5700557.

Keermann M, Kõks S, Reimann E, Prans E, Abram K, Kingo K. Transcriptional landscape of psoriasis identifies the involvement of IL36 and IL36RN. BMC Genomics 2015; 16(1): 322. doi: 10.1186/s12864-015-1508-2.

Reimann E, Kingo K, Karelson M, Reemann P, Loite U, Sulakatko H, et al. The mRNA expression profile of cytokines connected to the regulation of melanocyte functioning in vitiligo skin biopsy samples and peripheral blood mononuclear cells. Hum Immunol 2012; 73(4): 393-398. doi: 10.1016/j.humimm.2012.01.011.

Samadani AA, Nikbakhsh N, Fattahi S, Pourbagher R, Aghajanpour Mir SM, Mousavi Kani N, et al. RNA Extraction from Animal and Human's Cancerous Tissues: Does Tissue Matter? Int J Mol Cell Med 2015; 4(1): 54-59. PMID: 25815283.

Bird IM. Extraction of RNA from cells and tissue. Methods Mol Med. 2005;108:139-48. doi: 10.1385/1-59259-850-1:139. PMID: 16028681

Ding JY, Wang ZH, Zhang ZZ, Cui XR, Hong YY, Liu QQ. Effects of three IL-15 variants on NCI-H446 cell proliferation and expression of cell cycle regulatory molecules. Oncotarget. 2017;8(64):108108-108117. doi: 10.18632/oncotarget.22550. PMID: 29296227PMCID: PMC5746129

Li J, Xia Y, Liu T, Wang J, Dai W, Wang F, et al. Protective effects of astaxanthin on ConA-induced autoimmune hepatitis by the JNK/p-JNK pathway-mediated inhibition of autophagy and apoptosis. PLoS One. 2015 Mar 11;10(3):e0120440. doi: 10.1371/journal.pone.0120440. PMID: 25761053; PMCID: PMC4356569.

Chen F, Han B, Meng Y, Han Y, Liu B, Zhang B, et al. Ceruloplasmin correlates with immune infiltration and serves as a prognostic biomarker in breast cancer. Aging (Albany NY). 2021;13(16):20438-20467. doi: 10.18632/aging.203427. Epub 2021 Aug 19. PMID: 34413268; PMCID: PMC8436892.

Sun JH, Zhu XY, Dong TN, Zhang XH, Liu QQ, Li SQ, et al. An "up, no change, or down" system: Time-dependent expression of mRNAs in contused skeletal muscle of rats used for wound age estimation. Forensic Sci Int. 2017;272:104-110. doi: 10.1016/j.forsciint.2017.01.012. Epub 2017 Jan 17. PMID: 28129582.

Ortiz-Rey J, Suárez-Peñaranda J, San Miguel P, Muñoz J, Rodríguez-Calvo M, Concheiro L. Immunohistochemical analysis of P-Selectin as a possible marker of vitality in human cutaneous wounds. J Forensic Leg Med 2008; 15(6): 368-372. doi: 10.1016/j.jflm.2008.02.011.

Fieguth A, Feldbrügge H, Gerich T, Kleemann W, Tröger H. The time-dependent expression of fibronectin, MRP8, MRP14 and defensin in surgically treated human skin wounds. Forensic Sci Int 2003; 131(2-3):156-161. doi: 10.1016/s0379-0738 (02)00428-0.

Khalaf AA, Hassanen EI, Zaki AR, Tohamy AF, Ibrahim MA. Histopathological, immunohistochemical, and molecular studies for determination of wound age and vitality in rats. Int Wound J. 2019; 16(6): 1416-1425. doi: 10.1111/iwj.13206.

Grellner W. Time-dependent immunohistochemical detection of proinflammatory cytokines (IL-1β, IL-6, TNF-α) in human skin wounds. Forensic Sci Int. 2002;130:90–96.

Hernández-Cueto C, Girela E, Sweet DJ. Advances in the diagnosis of wound vitality: a review. Am J Forensic Med Pathol 2000; 21(1): 21-31. doi: 10.1097/00000433-200003000-00004.

Abd-Elhakim YM, Omran BHF, Ezzeldein SA, Ahmed AI, El-Sharkawy NI, Mohamed AA. Time-dependent expression of high-mobility group box-1 and toll-like receptors proteins as potential determinants of skin wound age in rats: Forensic implication. Int J Legal Med 2022; 136(6): 1781-1789. doi: 10.1007/s00414-022-02788-z.

Reimann E, Kingo K, Karelson M, Reemann P, Loite U, Sulakatko H, et al. The mRNA expression profile of cytokines connected to the regulation of melanocyte functioning in vitiligo skin biopsy samples and peripheral blood mononuclear cells. Hum Immunol 2012; 73(4): 393-398. doi: 10.1016/j.humimm.2012.01.011.

Liu CJ, Lien KY, Weng CY, Shin JW, Chang TY, Lee GB. Magnetic-bead-based microfluidic system for ribonucleic acid extraction and reverse transcription processes. Biomed Microdevices 2009; 11(2): 339-350. doi: 10.1007/s10544-008-9240-1.

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2023-06-16 — Updated on 2023-07-06

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