A rapid automated immunohistochemistry approach may help supplement intraoperative frozen-section hematoxylin and eosin staining in selected diagnostically ambiguous surgical pathology cases, according to a single-center validation study published in APMIS.
Researchers evaluated fast frozen rapid automated immunohistochemistry (FFRA-IHC) using the Q-Stain X Autostainer in tissue samples submitted for intraoperative frozen-section assessment at Rigshospitalet in Copenhagen, Denmark, from September 2023 to May 2024. Of 44 samples initially assessed, 42 were included in the final analysis after 2 exclusions.
The final data set included 37 tumors that could not be classified by frozen-section hematoxylin and eosin staining alone and 5 ambiguous resection margins. Final formalin-fixed paraffin-embedded analysis served as the reference standard.
Among the 37 previously unclassifiable tumors, FFRA-IHC allowed more specific classification in 25 cases, or 68%. The tumors included carcinomas, lymphomas, neuroendocrine tumors, benign lesions, 1 glioblastoma, and 1 atypical meningioma. Twelve tumors remained unclassified following the rapid immunohistochemistry workflow.
All 5 ambiguous resection margins were resolved with FFRA-IHC, although the margin analysis was based on a very small sample. In these cases, cytokeratin and CD45 staining demonstrated that atypical cells at the margins were lymphocytes or histiocytes rather than malignant epithelial cells.
The antibody panel included CK-AE, CK5, CK7, CD45, and synaptophysin. The researchers noted that the limited panel constrained diagnostic scope; MART-1, a melanoma marker, was not available for the system during the study.
The study also included representative cases illustrating potential clinical impact. In 1 case, FFRA-IHC identified an indeterminate neck lesion as carcinoma, prompting surgeons to search intraoperatively for a primary tumor while the patient remained anesthetized. In another, a maxillary sinus lesion initially suspected to represent carcinoma was rapidly reclassified as lymphoma, redirecting the patient toward hematologic evaluation rather than surgical management. Overall, the researchers estimated that FFRA-IHC potentially altered intraoperative management in approximately 12% of cases — a modest but clinically meaningful proportion given the stakes of real-time surgical decision-making.
Turnaround time for FFRA-IHC averaged approximately 21 minutes, with total time from specimen receipt to final intraoperative diagnosis of approximately 40 minutes. The department's quality benchmark for frozen-section reporting is 20 minutes from specimen receipt to pathologist report. The investigators noted that the initial frozen-section hematoxylin and eosin diagnosis was always communicated to the surgical team before FFRA-IHC was initiated, which allowed the extended workflow to remain clinically acceptable.
In a parallel operational comparison, the automated approach required less hands-on time and reduced exposure to hazardous chemicals compared with manual frozen-section immunohistochemistry. However, per-slide costs were higher, and the system required dedicated instrumentation.
The study was limited by its small sample size, single-center design, selected population of diagnostically challenging cases, and narrow antibody panel. The diagnostic impact assessment also relied on a semiquantitative scoring system developed by the investigators specifically for this study rather than a validated external instrument. The study did not evaluate patient outcomes, reoperation rates, or long-term clinical impact. Larger studies with expanded antibody panels are needed to better define the clinical role and broader applicability of FFRA-IHC in intraoperative pathology workflows — particularly whether the roughly 1-in-8 cases where management was potentially redirected translates into measurable differences in surgical and oncologic outcomes.
The researchers reported no conflicts of interest.
