Delayed fluorescence on indocyanine green angiography reflects impaired red blood cell perfusion: implications for intraoperative perfusion assessment in colorectal surgery.

Indocyanine green fluorescence angiography (ICG-FA) is widely used for intraoperative perfusion assessment in colorectal surgery. Although delayed fluorescence is commonly interpreted as impaired perfusion, its underlying hemodynamic characteristics remain unclear. A porcine model of mesenteric division was used to evaluate intestinal perfusion using both ICG-FA and Blue Laser Imaging-microcirculation (BLI-M), which enables direct visualization of red blood cell (RBC) flow. Perfusion boundaries, fluorescence dynamics, and microcirculatory flow were analyzed on both mucosal and serosal surfaces. A mucosal bridge model was created to assess the role of submucosal perfusion. In regions showing delayed fluorescence on ICG-FA, no detectable RBC flow was observed on either the mucosal or serosal surfaces using BLI-M. Despite this, fluorescence gradually increased and reached levels comparable to well-perfused regions. Quantitative analysis demonstrated prolonged time to fluorescence onset and reduced slope in delayed regions, while maximum fluorescence intensity was similar across regions. These findings suggest that delayed fluorescence reflects temporal delay rather than adequate perfusion, likely due to plasma-dominant flow or extremely low-velocity microcirculation. The perfusion boundary identified by ICG-FA corresponded to mucosal perfusion rather than serosal perfusion, indicating a layer-specific discrepancy in intestinal blood flow. Delayed fluorescence on ICG-FA may represent regions with insufficient effective RBC perfusion despite preserved fluorescence intensity. Accurate identification of the initial perfusion boundary is essential, as reliance on delayed fluorescence may lead to inappropriate transection line selection and increased risk of anastomotic leakage. To our knowledge, this is the first study to provide direct experimental evidence elucidating the mechanism underlying delayed fluorescence observed in ICG-FA.