During the harvest and subsequent processing of blood vessels for allogeneic transplantation, DNA damage can occur. A suitable method for quantifying this damage may be the comet assay, also known as the single cell gel electrophoresis assay, which is used to measure DNA damage at the single-cell level. We evaluated the key indicators of muscle cell DNA damage in the tunica media of arteries by using the comet assay with four groups: (1) cryopreserved grafts thawed slowly, (2) cryopreserved grafts thawed rapidly, (3) cold-preserved "fresh" grafts harvested as part of a multiorgan procurement, and (4) cadaveric arterial graft harvested at autopsy. Kruskal-Wallis multiple-comparison testing identified a statistically significant difference between rapidly thawed grafts and multiorgan harvested fresh grafts. Evans' correlation criterion indicated a moderately strong correlation between the percentage of DNA in the comet head and warm ischemia duration in the group of slowly thawed grafts, and between the percentage of DNA in the comet head and cold ischemia duration in the group of cadaveric grafts. No association of come assay parameters with age was demonstrated. Comet assay showed that the cryopreservation and storage processes described in this study did not lower the DNA content in comparison with fresh grafts collected during multiorgan harvests in operating rooms and that DNA content was not influenced by the type of thawing protocol.

2nd Dept of Surgery Dept of Cardiovascular Surgery 1st Faculty of Medicine Charles University and General University Hospital Prague Czech Republic

Department of Cardio Vascular Surgery WIGEV Clinic Floridsdorf and Karl Landsteiner Institute for Cardio Vascular Research Vienna Austria

Department of Histology and Embryology Faculty of Medicine in Hradec Králové Charles University Hradec Králové Czech Republic

Department of Medical Biophysics Faculty of Medicine in Hradec Králové Charles University Hradec Králové Czech Republic

Department of Transplantation Surgery Institute for Clinical and Experimental Medicine Prague and 1st Faculty of Medcine Charles University Prague Czech Republic

Institute of Forensic Medicine and Toxicology General University Hospital and 1st Faculty of Medicine Charles University Prague Czech Republic

Technology Centre of the Czech Academy of Sciences Department of Strategic Studies Prague Czech Republic

Tissue Bank University Hospital Hradec Králové Hradec Králové Czech Republic

Zobrazit více v PubMed

Gross RE, Bill AH Jr, Peirce EC Jr. Methods for preservation and transplantation of arterial grafts; observations on arterial grafts in dogs; report of transplantation of preserved arterial grafts in nine human cases. Surg Gynecol Obstet. 1949;88(6):689–701. PubMed

Dubost C, Allary M, Oeconomos Null. Aneurysm of the abdominal aorta treated by resection and graft. Arch Mal Coeur Vaiss. 1951;44(9):848–51. PubMed

Szilagyi DE, McDonald RT, Smith RF, Whitcomb JG. Biologic fate of human arterial homografts. AMA Arch Surg. 1957;75(4):506–27; discussion 527-9. doi: 10.1001/archsurg.1957.01280160016003 PubMed DOI

Szilagyi DE, Smith RF, Elliott JP, Allen HM. Long-term behavior of a dacron arterial substitute: clinical, roentgenologic and histologic correlations. Ann Surg. 1965;162(3):453–77. doi: 10.1097/00000658-196509000-00015 PubMed DOI PMC

Szilagyi DE, Rodriguez FJ, Smith RF, Elliott JP. Late fate of arterial allografts. Observations 6 to 15 years after implantation. Arch Surg. 1970;101(6):721–33. doi: 10.1001/archsurg.1970.01340300077014 PubMed DOI

Lehalle B, Geschier C, Fiévé G, Stoltz JF. Early rupture and degeneration of cryopreserved arterial allografts. J Vasc Surg. 1997;25(4):751–2. doi: 10.1016/s0741-5214(97)70305-7 PubMed DOI

Pegg DE, Wusteman MC, Boylan S. Fractures in cryopreserved elastic arteries. Cryobiology. 1997;34(2):183–92. doi: 10.1006/cryo.1996.1997 PubMed DOI

Bisdas T, Bredt M, Pichlmaier M, Aper T, Wilhelmi M, Bisdas S, et al. Eight-year experience with cryopreserved arterial homografts for the in situ reconstruction of abdominal aortic infections. J Vasc Surg. 2010;52(2):323–30. doi: 10.1016/j.jvs.2010.02.277 PubMed DOI

Vogt PR. Arterial allografts in treating aortic graft infections: something old, something new. Semin Vasc Surg. 2011;24(4):227–33. doi: 10.1053/j.semvascsurg.2011.10.008 PubMed DOI

Kieffer E, Gomes D, Chiche L, Fléron M-H, Koskas F, Bahnini A. Allograft replacement for infrarenal aortic graft infection: early and late results in 179 patients. J Vasc Surg. 2004;39(5):1009–17. doi: 10.1016/j.jvs.2003.12.040 PubMed DOI

Spunda R, Hruby J, Mericka P, Mlcek M, Pecha O, Splith K, et al. Immunosuppressive protocols with tacrolimus after cryopreserved aortal allotransplantation in rats. PLoS One. 2018;13(8):e0201984. doi: 10.1371/journal.pone.0201984 PubMed DOI PMC

Jashari R, Van Hoeck B, Ngakam R, Goffin Y, Fan Y. Banking of cryopreserved arterial allografts in Europe: 20 years of operation in the European Homograft Bank (EHB) in Brussels. Cell Tissue Bank. 2013;14(4):589–99. PubMed

Johnson S, Rabinovitch P. Ex vivo imaging of excised tissue using vital dyes and confocal microscopy. Curr Protoc Cytom. 2012. PubMed PMC

Doonan F, Cotter TG. Morphological assessment of apoptosis. Methods. 2008;44(3):200–4. doi: 10.1016/j.ymeth.2007.11.006 PubMed DOI

Krysko DV, Vanden Berghe T, D’Herde K, Vandenabeele P. Apoptosis and necrosis: detection, discrimination and phagocytosis. Methods. 2008;44(3):205–21. doi: 10.1016/j.ymeth.2007.12.001 PubMed DOI

Špaček M, Měřička P, Janoušek L, Dalecká M, Benda A, Krs O, et al. Comparison of different thawing protocols in human cryopreserved venous grafts. Ann Vasc Surg. 2020;64:347–54. doi: 10.1016/j.avsg.2019.11.026 PubMed DOI

Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol. 2007;35(4):495–516. doi: 10.1080/01926230701320337 PubMed DOI PMC

Archana M, Bastian, Yogesh TL, Kumaraswamy KL. Various methods available for detection of apoptotic cells--a review. Indian J Cancer. 2013;50(3):274–83. doi: 10.4103/0019-509X.118720 PubMed DOI

Huerta S, Goulet EJ, Huerta-Yepez S, Livingston EH. Screening and detection of apoptosis. J Surg Res. 2007;139(1):143–56. doi: 10.1016/j.jss.2006.07.034 PubMed DOI

Sakamuru S, Li X, Attene-Ramos MS, Huang R, Lu J, Shou L, et al. Application of a homogenous membrane potential assay to assess mitochondrial function. Physiol Genomics. 2012;44(9):495–503. doi: 10.1152/physiolgenomics.00161.2011 PubMed DOI PMC

McIlwain DR, Berger T, Mak TW. Caspase functions in cell death and disease. Cold Spring Harb Perspect Biol. 2013;5(4):a008656. doi: 10.1101/cshperspect.a008656 PubMed DOI PMC

Clementi E, Garajova Z, Markkanen E. Measuring DNA damage using the alkaline comet assay in cultured cells. Bio Protoc. 2021;11(16):e4119. doi: 10.21769/BioProtoc.4119 PubMed DOI PMC

Collins AR. Measuring oxidative damage to DNA and its repair with the comet assay. Biochim Biophys Acta. 2014;1840(2):794–800. doi: 10.1016/j.bbagen.2013.04.022 PubMed DOI

Kuchařová M, Hronek M, Rybáková K, Zadák Z, Štětina R, Josková V. Comet assay and its use for evaluating oxidative DNA damage in some pathological states. Physiol Res. 2019;:1–15. PubMed

Collins AR. The comet assay for DNA damage and repair: principles, applications, and limitations. Mol Biotechnol. 2004;26(3):249–61. doi: 10.1385/MB:26:3:249 PubMed DOI

Pilin A, Mazura I, Lainkova R, Salmay M, Měřička P, Pecha O, et al. Viability of human arterial grafts monitored by comet assay. Physiol Res. 2024;73(2):217–25. doi: 10.33549/physiolres.935055 PubMed DOI PMC

Piperakis SM. Comet assay: a brief history. Cell Biol Toxicol. 2009;25(1):1–3. doi: 10.1007/s10565-008-9081-y PubMed DOI

Evans RH. An analysis of criterion variable reliability in conjoint analysis. Percept Mot Skills. 1996;82(3):988–90. doi: 10.2466/pms.1996.82.3.988 DOI

Olive PL. The comet assay. An overview of techniques. Methods Mol Biol. 2002;203:179–94. doi: 10.1385/1-59259-179-5:179 PubMed DOI

Špaček M, Měřička P, Janoušek L, Štádler P, Adamec M, Vlachovský R, et al. Organization model for allotransplantations of cryopreserved vascular grafts in Czech Republic. Cell Tissue Bank. 2018;19(3):437–45. doi: 10.1007/s10561-018-9691-4 PubMed DOI

Špaček M, Měřička P, Janoušek L, Štádler P, Adamec M, Vlachovský R, et al. Current vascular allograft procurement, cryopreservation and transplantation techniques in the Czech Republic. Adv Clin Exp Med. 2019;28(4):529–34. doi: 10.17219/acem/90037 PubMed DOI

Měřička P, Janoušek L, Benda A, Lainková R, Sabó J, Dalecká M, et al. Cell viability assessment using fluorescence vital dyes and confocal microscopy in evaluating freezing and thawing protocols used in cryopreservation of allogeneic venous grafts. Int J Mol Sci. 2021;22(19):10653. doi: 10.3390/ijms221910653 PubMed DOI PMC

Kumaravel TS, Jha AN. Reliable comet assay measurements for detecting DNA damage induced by ionising radiation and chemicals. Mutat Res. 2006;605(1–2):7–16. doi: 10.1016/j.mrgentox.2006.03.002 PubMed DOI

Igl BW. Statistical analysis of the comet assay. In: Promoting statistical insight toxicology. Berlin. 2018.

Ministerstvo zdravotnictví ČR. Vyhláška o stanovení bližších požadavků pro zajištění jakosti a bezpečnosti lidských tkání a buněk určených k použití u člověka. 2017.

European Directorate for the Quality of Medicines Council of Europe. Guide to the quality and safety of tissues and cells for human application. 5th ed. Strasbourg, France: Council of Europe; 2022.

Měřička P, Špaček M, Janoušek L, Dvořáček L, Štádler P, Vlachovský R, et al. Cryopreservation of vascular grafts for clinical use – Retrospective analysis of pre-freezing factors with potential impact on the quality and safety of vascular transplantations. Cryobiology. 2015;71(3):546–7. doi: 10.1016/j.cryobiol.2015.10.041 DOI

Jashari R, Bouzet V, Alcaraz Blanco MJ, Oleffe A, Lecocq E, Mastrobuoni S. Vascular allografts for clinical application in Europe: assessment of 30 years of experience with vascular tissue banking in Brussels. Cell Tissue Bank. 2023;24(3):613–25. PubMed PMC

Acker JP, Bondarovych M, Brunotte R, Buriak IA, Fuller BJ, Glasmacher B, et al. Preservation and storage of cells for therapy: current applications and protocols. In: Reference series in biomedical engineering. Springer International Publishing; 2022. 1–69. doi: 10.1007/978-3-319-37076-7_68-1 DOI

Fellmer PT, Matia I, Jonas S. Arterial allografts in vascular surgery--best choice in cases of aortic graft infection?!. Zentralbl Chir. 2013;138(5):530–5. doi: 10.1055/s-0032-1328624 PubMed DOI

Hruby J, Spunda R, Mericka P, Mlcek M, Pecha O, Splith K, et al. Influence of the new standardized clinical cryopreservation/slow thawing protocol on immunogenicity of arterial allografts in rats. PLoS One. 2020;15(3):e0230234. doi: 10.1371/journal.pone.0230234 PubMed DOI PMC