Ex vivo fluorescence confocal microscopy combined with rapid immunofluorescence for intraoperative intestinal mapping in near-total intestinal aganglionosis: a pediatric proof-of-concept case report

Introduction: In Hirschsprung disease (HD), intraoperative identification of ganglionated bowel remains central to surgical planning, particularly in extensive or near-total intestinal aganglionosis (nTIA), where bowel preservation is critical. Frozen-section histopathology remains the intraoperative standard. Ex-vivo fluorescence confocal microscopy (FCM) provides microscopic assessment of fresh tissue, while rapid immunofluorescence (rIF) has the potential to add targeted visualization of enteric neural structures. We describe a single pediatric proof-of-concept case in which FCM combined with selected rIF was integrated into intraoperative intestinal mapping.Case-report: A full-term newborn, diagnosed at birth with nTIA, underwent tube stoma formation at 40 cm from the Treitz ligament without bowel resection. At 4-years, surgery was scheduled to remove aganglionic segments and lengthen the remaining bowel. During surgery the small bowel segment proximal to the ostomy measured 70 cm. Twenty-four full-thickness biopsies were collected every 20 cm from sigmoid colon to presumed ganglionic areas at stoma level. All samples underwent FCM for morphological assessment of fresh tissue. rIF was applied to four selected samples, two from presumed ganglionated bowel and two from presumed aganglionic bowel, as an adjunct to enhance visualization of S100B-positive neural and glial structures. Fresh tissue was incubated for 30 min with a primary anti-S100B antibody followed by an Alexa Fluor 647-conjugated secondary antibody, then rinsed and counterstained with acridine orange. Under fluorescence laser visualization ganglion cells were identified in functional segments while hypertrophic nerve fibers were detected in aganglionic regions. Surgical planning relied on integrated intraoperative assessment, with final confirmation by conventional histopathology. The simultaneous application of a longitudinal intestinal lengthening (LILT) ensured maximization of functional bowel.Conclusion: This single-case experience demonstrates the technical feasibility of integrating ex vivo FCM and rIF into intraoperative intestinal mapping for complex Hirschsprung disease. The approach should be regarded as exploratory and complementary to established histopathological assessment.