## 4 Discussion
The typical CH-50 assay is useful for human, bovine, porcine, and rabbit sera; however, the sensitivity is too low to detect complement activation after contact with the material. Another functional method is complementing production of C3- or C5-converting enzymes as measured via substrate conversion assays. The ISO 10993-4 standard (2017) provides information about complement testing of medical materials and devices. No standard blood or blood product is currently used for complement testing, and when serum and plasma are used, they must be carefully selected so as not to reduce or increase complement activation [1]. In this study, to select standard blood and blood products used for complement testing, human and porcine sera were reacted with the material using the direct contact method, and then complement activation was assayed. By observing complement activation with human serum and one negative and two positive control candidate materials as the test materials, negative and positive control candidate materials could be effectively distinguished. In contrast, they were difficult to distinguish in porcine serum. Tests with strong activators, such as CVF, confirmed that it works under specific conditions. Complement activation was measured in CVF, a potent activator, and human and porcine sera. Complement activation was significantly increased in human serum but showed a basal level of activation in porcine serum. This indicates significant differences between humans and porcine in their potential to activate the complement system [28; 29]. Porcine ex vivo and implantation models are the preferred preclinical models for hemocompatibility evaluation of blood-contacting medical devices owing to their anatomical similarity to human organs [30; 31; 32]. Despite porcine blood exhibiting high sensitivity in the coagulation reaction after contact with biomaterials, the result showing a basal reaction in the activity of the complement system must be considered in the design of the preclinical test model and interpretation of the results. The results revealed that the normalized values of PTT in substance-reacted plasma exhibited similarities regardless of the animal species, and the blood coagulation potential was confirmed in various animal species. In addition, the results of complement activation for a material indicate that the reactivity differs between species and provide important clues in the interpretation of the results of preclinical testing of blood-contacting medical devices and in the design of experiments. In addition, the selection of an appropriate animal model for thrombosis evaluation indicates the potential to substitute human blood and may correlate with in vivo and clinical thrombosis data for thrombosis evaluation. [PERSON] performed most of the in vitro experiments and wrote the original draft. [PERSON], [PERSON], [PERSON] and [PERSON] advised the experiment. [PERSON] and [PERSON] analyzed the final data and edited the final manuscript. All authors have read and agreed to the published version of the manuscript. This research was supported by a grant (21174 MFDS242) from the Ministry of Food and Drug Safety in 2022. **Institutional Review Board Statement:** The animal study protocol was approved by the Institutional Review Board of Futuristic Animal Resource and Research Center (FARRC-220401 and date of approval 1 April 2022), the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Institutional Animal Care and Use Committee (NPRC-220401 and date of approval 1 April 2022), and the Korea Testing Certification Institutional Animal Care and Use Committee (Protocol-ETCS-02 and date of approval 14 May 2021). **Informed Consent Statement:** Not applicable. **Data Availability Statement:** The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author. **Acknowledgments:** We thank everyone who helped. **Conflicts of Interest:** The authors declare no conflicts of interest.