131 Quantitative analysis of intestinal perfusion with ICG and MB Limitations There are some limitations in this animal study. The small sample size is a notable limitation. However, to ensure the feasibility of our hypothesis and adhere to the 3R principle (i.e., replace, reduce, refine) in animal research 27, the number of animals used in the study was considered adequate. Despite this small sample size, we believe that the correlations we present are sufficiently illustrative. Each pig underwent 5 measurements, resulting in a total of 20 measurements for each correlation analysis, which is sufficient for a Spearman correlation. Another limitation is that, although laparoscopic procedures are the norm for most elective abdominal clinical procedures, we used an open camera system in this study due to logistical constraints. Fortunately, there is a commercially available laparoscopic variant of the camera system used in our study. Additionally, while anastomotic perfusion is commonly required during colorectal resection and anastomosis creation, we used small bowel loops in our experiment. This decision was based on the challenges posed by the fixed and spiral orientation of a pig’s colon, making the small bowel a more suitable choice for illustrative purposes. It is essential to acknowledge that it differs from human colonic tissue, underscoring the need for human studies to provide crucial insights. Consequently, it is critical to interpret the current results cautiously, and human studies are necessary to assess the reproducibility of our findings in a clinical context. CONCLUSION To conclude, we successfully performed a quantification analysis of a commercially available NIRF imaging system in this study. We demonstrated a significant negative correlation of ingress values of MB and ICG fluorescence quantification analysis with local lactate levels. This validates the potential to use MB for bowel perfusion assessment besides the wellknown and widely used ICG. Further human studies are necessary to translate our findings to clinical applications. ACKNOWLEDGMENTS AND STATEMENTS The authors would like to thank the staff of the animal laboratory of the Maastricht University Medical Center (MUMC+, Maastricht, The Netherlands) for their help during the animal study. We would also like to thank Bjorn Winkens, who was involved as a professional statistician to ensure an appropriate statistical analysis, as well as Basma Benhaddou and Guy Temporal for their proofreading assistance. This experiment was graciously funded by the European Association for Endoscopic Surgery and other Interventional Techniques (EAES). QUEST SPECTRUM® (Quest Medical Imaging, Middenmeer, The Netherlands) provided the NIRF imaging system. 6
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