Testing the hemocompatibility of medical devices after their interaction with blood entails the need to evaluate the activation of blood elements and the degree of their coagulation and adhesion to the device surface. One possible way to achieve this is to use scanning electron microscopy (SEM). The aim was to develop a novel SEM-based method to assess the thrombogenic potential of medical devices and their adhesiveness to blood cells. As a part of this task, also find a convenient procedure of efficient and non-destructive sample fixation for SEM while reducing the use of highly toxic substances and shortening the fixation time. A polymeric surgical mesh was exposed to blood so that blood elements adhered to its surface. Such prepared samples were then chemically fixed for a subsequent SEM measurement; a number of fixation procedures were tested to find the optimal one. The fixation results were evaluated from SEM images, and the degree of blood elements' adhesion was determined from the images using ImageJ software. The best fixation was achieved with the May-Grünwald solution, which is less toxic than chemicals traditionally used. Moreover, manipulation with highly toxic osmium tetroxide can be avoided in the proposed procedure. A convenient methodology for SEM image analysis has been developed too, enabling to quantitatively evaluate the interaction of blood with the surfaces of various medical devices. Our method replaces the subjective assessment of surface coverage with a better-defined procedure, thus offering more precise and reliable results.

Department of Biology Faculty of Science University of Hradec Králové Rokitanského 62 Hradec Králové 500 03 Czech Republic

Department of Physics Faculty of Science University of Hradec Králové Rokitanského 62 Hradec Králové 500 03 Czech Republic

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Kuchinka J, Willems C, Telyshev DV, Groth T. Control of blood coagulation by hemocompatible material surfaces—A review. Bioengineering. 2021;8:215. PubMed PMC

Elvers FL, Stamouli M, Adelmeijer J, Jeyanesan D, Bernal W, Maas C, Patel VC, Lisman T. In vivo generation of thrombin in patients with liver disease without apparent evidence of activation of the intrinsic or extrinsic pathway of coagulation. J. Thromb. Haemost. 2023;21:2078–2088. PubMed

Ehlerding G, Erlenkötter A, Gauly A, Griesshaber B, Kennedy J, Rauber L, Ries W, Schmidt-Gürtler H, Stauss-Grabo M, Wagner S, Zawada AM, Zschätzsch S, Kempkes-Koch M. Performance and hemocompatibility of a novel polysulfone dialyzer: A randomized controlled Trial. Kidney. 2021;2:937–947. PubMed PMC

Roberts TR, Seekell RP, Zang Y, Harea G, Zhang Z, Batchinsky AI. In vitro hemocompatibility screening of a slippery liquid impregnated surface coating for extracorporeal organ support applications. Perfusion. 2022;39(1):76–84. PubMed

ISO. EN ISO 10993-1:2018 Biological Evaluation of Medical Devices—Part 1: Evaluation and Testing within a Risk Management Process (2018).

van Oeveren W, Tielliu IF, de Hart J. Comparison of modified chandler, roller pump, and ball valve circulation models for in vitro testing in high blood flow conditions: Application in thrombogenicity testing of different materials for vascular applications. Int. J. Biomater. 2012;2012:673163. PubMed PMC

Schatz T, Howard S, Porter D, Grove K, Smith ME, Chen Y. Improved method for the ASTM platelet and leukocyte assay: Use of minimal heparinization in a screening test for hemocompatibility of blood-contacting medical devices. J. Med. Device. 2019;13(1):011004.

Paavilainen L, Edvinsson Å, Asplund A, Hober S, Kampf C, Pontén F, Wester K. The impact of tissue fixatives on morphology and antibody-based protein profiling in tissues and cells. J. Histochem. Cytochem. 2010;58:237–246. PubMed PMC

Ichikawa T, Wang D, Miyazawa K, Miyata K, Oshima M, Fukuma T. Chemical fixation creates nanoscale clusters on the cell surface by aggregating membrane proteins. Commun. Biol. 2022;5:487. PubMed PMC

Huebinger J, Spindler J, Holl KJ, Koos B. Quantification of protein mobility and associated reshuffling of cytoplasm during chemical fixation. Sci Rep. 2018;8:17756. PubMed PMC

Eltoum I, Fredenburgh J, Myers RB, Grizzle WE. Introduction to the theory and practice of fixation of tissues. J. Histotechnol. 2001;24:173–190.

Xu M, Liu J, Sun J, Xu X, Hu Y, Liu B. Optical microscopy and electron microscopy for the morphological evaluation of tendons: A mini review. Orthop Surg. 2020;12:366–371. PubMed PMC

Gauer JS, Duval C, Xu R-G, Macrae FL, McPherson HR, Tiede C, Tomlinson D, Watson SP, Ariëns RAS. Fibrin-glycoprotein VI interaction increases platelet procoagulant activity and impacts clot structure. J. Thromb. Haemost. 2023;21:667–681. PubMed

Ul-Hamid A. A beginners’ guide to scanning electron microscopy. 1. Cham: Springer; 2018.

Migneault I, Dartiguenave C, Bertrand MJ, Waldron KC. Glutaraldehyde: Behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking. Biotechniques. 2004;37:798–802. PubMed

Tayri-Wilk T, Slavin M, Zamel J, Blass A, Cohen S, Motzik A, Sun X, Shalev DE, Ram O, Kalisman N. Mass spectrometry reveals the chemistry of formaldehyde cross-linking in structured proteins. Nat. Commun. 2020;11:3128. PubMed PMC

Modenez IA, Sastre D, Moraes FC, Netto CM. Influence of glutaraldehyde cross-linking modes on the recyclability of immobilized Lipase B from candida Antarctica for transesterification of soy bean oil. Molecules. 2018;23:2230. PubMed PMC

Thumtecho S, Sriapha C, Tongpoo A, Udomsubpayakul U, Wananukul W, Trakulsrichai S. Poisoning of glutaraldehyde-containing products: Clinical characteristics and outcomes. Clin. Toxicol. (Phila) 2021;59:480–487. PubMed

Matei A, Puscas C, Patrascu I, Lehene M, Ziebro J, Scurtu F, Baia M, Porumb D, Totos R, Silaghi-Dumitrescu R. Stability of glutaraldehyde in biocide compositions. Int. J. Mol. Sci. 2020;21:3372. PubMed PMC

Hopwood D. Some aspects of fixation with glutaraldehyde. A biochemical and histochemical comparison of the effects of formaldehyde and glutaraldehyde fixation on various enzymes and glycogen, with a note on penetration of glutaraldehyde into liver. J. Anat. 1967;101:83–92. PubMed PMC

National Center for Biotechnology Information. PubChem Compound Summary for CID 712, Formaldehyde (2023).

National Center for Biotechnology Information. PubChem Compound Summary for CID 3485, Glutaraldehyde (2023).

Wilson S, Law SP, McEwan NR, Wright R, Macaskill JS. Development of a fixative protocol using formaldehyde and gluteraldehyde for preservation of microbial art on agar plates. J. Appl. Microbiol. 2022;133:665–672. PubMed PMC

Al Shehadat S, Gorduysus MO, Hamid SSA, Abdullah NA, Samsudin AR, Ahmad A. Optimization of scanning electron microscope technique for amniotic membrane investigation: A preliminary study. Eur. J. Dent. 2018;12:574–578. PubMed PMC

Hopwood D. Fixatives and fixation: A review. Histochem. J. 1969;1:323–360. PubMed

Buesa RJ. Histology without formalin? Ann. Diagn. Pathol. 2008;12:387–396. PubMed

Suvarna, S. K., Layton, C., Bancroft J. D. (eds) Bancroft’s Theory and Practice of Histological Techniques 8th edn (Elsevier, 2019).

Campbell BC, Paez-Segala MG, Looger LL, Petsko GA, Liu CF. Chemically stable fluorescent proteins for advanced microscopy. Nat. Methods. 2022;19:1612–1621. PubMed PMC

National Center for Biotechnology Information. PubChem Compound Summary for CID 30318, Osmium tetroxide. 2023.

Koga D, Kusimi S, Watanabe T. Optimizing the reaction temperature to facilitate an efficient osmium maceration procedure. Biomed. Res. 2020;41:161–168. PubMed

de Lima-Faria JM, Guimarães LN, da Silva VC, da Costa SI, Fernandes MN, Martinez DST, de Sabóia-Morais SMT. Distribution and behavior of lipid droplets in hepatic cells analyzed by variations of citochemical technique and scanning electron microscopy. MethodsX. 2022;9:101769. PubMed PMC

Graham L, Orenstein JM. Processing tissue and cells for transmission electron microscopy in diagnostic pathology and research. Nat. Protoc. 2007;2:2439–2450. PubMed PMC

Troiano NW, Ciovacco WA, Kacena MA. The effects of fixation and dehydration on the histological quality of undecalcified murine bone specimens embedded in methylmethacrylate. J. Histotechnol. 2009;32:27–31. PubMed PMC

Golding CG, Lamboo LL, Beniac DR, Booth TF. The scanning electron microscope in microbiology and diagnosis of infectious disease. Sci. Rep. 2016;6:26516. PubMed PMC

Nikara S, Ahmadi E, Nia AA. Effects of different preparation techniques on the microstructural features of biological materials for scanning electron microscopy. J. Agric. Food Res. 2020;2:100036.

Bhattacharya R, Saha S, Kostina O, Muravnik L, Mitra A. Replacing critical point drying with a low-cost chemical drying provides comparable surface image quality of glandular trichomes from leaves of Millingtonia hortensis L. f. in scanning electron micrograph. Appl. Microsc. 2020;50:15. PubMed PMC

Sharma R, Digaum JL, West H, Schwarz CM, Kuebler SM. Gentle method for removing metal and restoring function after scanning electron microscopy. J. Micro. Nanopattern. Mater. Metrol. 2021;20:023601.

Andrsova, Z., Kejzlar, P., Volesky, L., Petru, M. The effect of deposition of metal charge compensation coating on surface morfology of samples for SEM. In Proceedings 9th International Conference on Nanomaterials—Research & Application 848–853 (TANGER Ltd., Ostrava, 2018).

Bender M, Palankar R. Platelet shape changes during thrombus formation: Role of actin-based protrusions. Hamostaseologie. 2021;41:14–21. PubMed

Application Booklet: LeicaEM CPD300 Automated Critical Point Dryer. Leica Microsystems (2014).

Piaton E, Fabre M, Goubin-Versini I, Bretz-Grenier M-F, Courtade-Saïdi M, Vincent S, Belleannée G, Thivolet F, Boutonnat J, Debaque H, Fleury-Feith J, Vielh P, Cochand-Priollet B, Egelé C, Bellocq J-P, Michiels J-F. Technical recommendations and best practice guidelines for May–Grünwald-Giemsa staining: Literature review and insights from the quality assurance. Ann. Pathol. 2015;35:294–305. PubMed

Montanaro J, Gruber D, Leisch N. Improved ultrastructure of marine invertebrates using non-toxic buffers. PeerJ. 2016;4:e1860. PubMed PMC

Seyferth D. Cadet’s fuming arsenical liquid and the cacodyl compounds of bunsen. Organometallics. 2001;20:1488–1498.

Melo LL, Vaz AR, Salvadori MC, Cattani M. Grain sizes and surface roughness in platinum and gold thin films. J. Metastable Nanocrystal. Mater. 2004;20–21:623–628.

Nguyen TH, Palankar R, Bui VC, Medvedev N, Greinacher A, Delcea M. Rupture forces among human blood platelets at different degrees of activation. Sci. Rep. 2016;6:25402. PubMed PMC

Helenius J, Heisenberg CP, Gaub HE, Muller DJ. Single-cell force spectroscopy. J. Cell Sci. 2008;121:1785–1791. PubMed

Koltai K, Kesmarky G, Feher G, Tibold A, Toth K. Platelet aggregometry testing: Molecular mechanisms, techniques and clinical implications. Int. J. Mol. Sci. 2017;18:1803. PubMed PMC

Vanslembrouck B, Chen JH, Larabell C, van Hengel J. Microscopic visualization of cell-cell adhesion complexes at micro and nanoscale. Front. Cell Dev. Biol. 2022;10:819534. PubMed PMC