Standardising Immunocytochemistry on Breast Cytology: A Formalin Post‐Fixation Protocol Achieves High Concordance With Immunohistochemistry for Oestrogen Receptor, Progesterone Receptor, and Human Epidermal Growth Factor Receptor 2

INTRODUCTION: Standardisation of immunocytochemistry (ICC) on cytological specimens remains a major unmet need. Variability in fixation, antigen retrieval and staining platforms has limited reproducibility. In particular, reliable evaluation of oestrogen receptor (ER), progesterone receptor (PgR) and human epidermal growth factor receptor 2 (HER2) expression in cytology has been challenging due to heterogeneous pre-analytical conditions. We hypothesised that the introduction of a brief formalin post-fixation step would enable ICC on smear cytology to align more closely with the analytical conditions of immunohistochemistry (IHC). METHODS: A total of 120 breast cancer specimens from surgical resections were analysed. Smear cytology specimens were prepared using fine-needle aspiration or touch imprints, which were initially ethanol-fixed and stained with Papanicolaou stain. Slides with adequate tumour cellularity were subjected to 10% neutral-buffered formalin post-fixation before ICC was performed using the Ventana Benchmark ULTRA platform. ER, PgR and HER2 immunostaining on cytology slides were performed with the same standard protocols applied to formalin-fixed paraffin-embedded tissue IHC. RESULTS: The overall concordance between cytology ICC and tissue IHC was 96.7% for ER (κ: 0.893), 92.5% for PgR (κ: 0.803) and 80.0% for HER2 (weighted κ: 0.794). Most discordant cases were cytology-negative and tissue-positive for hormone receptors, with low positivity percentages in the tissue specimens. HER2 discordance predominantly occurred in cases with intermediate HER2 scores. All IHC 3+ cases were fully concordant. CONCLUSION: Formalin post-fixation enabled breast cytology ICC to be performed under conditions comparable to those of IHC, resulting in high concordance for breast cancer biomarkers. This simple pre-analytical step offers a practical and reproducible method for methodological standardisation.