Validation of ancillary procedures on formalin liquid fixed aspiration cytologic samples: from minimum to maximum

We sought to present an alternative liquid-based cytologic (aLBC) and an agarose-based cell block (CB) preparation method for formalin-fixed fine-needle aspirations (FNAs), using breast cancer as a model, and to perform a validation procedure for immunohistochemical (IHC) assays.

Between 2024 and 2025, 18 breast cancer FNA cases were collected and processed into agarose-based CB, and by applying the dropped reaspirated monolayer dried preparation method, into aLBC. Matched formalin-fixed, paraffin-embedded surgical specimens from the same patients served as gold-standard controls. Ten immunomarkers were assessed using validated IHC protocols. Human epidermal growth factor receptor 2 (HER2) status was evaluated by both IHC and dual-probe fluorescence in situ hybridization (FISH) according to 2023 American Society of Clinical Oncology/College of American Pathologists guidelines. Staining intensity, percentage of positive cells, and marker concordance were analyzed across all specimen types. Diagnostic performance metrics were calculated, and intermethod agreement was assessed using Cohen's κ coefficient (κ > 0.75 considered acceptable).

The sensitivity, specificity, and either negative or positive predictive values and the accuracy values were 100% at all the tested immunostains and at the HER2 FISH assays.

Accurate diagnosis from scant FNA material is an increasing demand in cytopathology. The validated aLBC and CB preparation methods proved to be cost-effective, efficient for ancillary testing, and reliable for IHC, even in low-cellularity samples.