Rapid Automated Immunohistochemistry on Frozen Sections Enables Real-Time Surgical Pathology Decisions

Intraoperative frozen section (FS) analysis is critical in surgical pathology, but conventional hematoxylin and eosin (H&E) staining has limitations in poorly differentiated neoplasms and resection margins. Immunohistochemistry (IHC) provides higher diagnostic specificity, yet has traditionally been too time-consuming for intraoperative urgency. This study aimed to optimize, validate, and evaluate Fast Frozen Rapid Automated Immunohistochemistry (FFRA-IHC) using the Q-Stain X Autostainer to improve surgical diagnostics. After optimization with antibodies CKAE, CK5, CK7, CD45, and Synaptophysin, 44 tissue samples from patients undergoing surgery at Rigshospitalet, Copenhagen, were analyzed by FS H&E, FFRA-IHC, and standard formalin-fixed paraffin-embedded (FFPE) methods. Automated FFRA-IHC showed high diagnostic accuracy and supported immediate clinical decision-making: of 37 tumors not classifiable by FS H&E alone, 25 (68%) were classified into specific tumor types, and all five ambiguous resection margins were resolved. A cost–benefit analysis indicated efficiency gains with reduced hands-on time compared to manual IHC. In conclusion, FFRA-IHC demonstrated promising results for enhancing intraoperative diagnostics, leading to its implementation in the daily workflow at our department. Future studies should expand antibody panels and assess the broader clinical impact to further improve intra- and perioperative care.