Practical issues related to immunocytochemistry on cytological smears: Tips and recommendations

Objective Immunocytochemistry (ICC) is essential for enhancing diagnostic accuracy and identifying markers for diagnosis, prognosis and targeted therapies. While cell blocks (CBs) are preferred for standardization and optimized staining, cytological smears are an alternative when CBs are unavailable. However, the literature on ICC protocols for smears is sparse. This review addresses preparation, fixation and protocols for nuclear and cytoplasmic antibodies on smears, drawing from our laboratory's experience. Methods We reviewed procedures for ICC on cytological smears using existing literature and practical insights from our laboratory. Results Commercially available antibodies were found to be reliable for ICC on smears if specimens are properly prepared and fixed. Protocols developed in our laboratory maintained antigenicity and provided clear staining results. Conclusions Although ICC on CBs is the gold standard for standardization, cytological smears are a viable alternative when CBs are unavailable. Success in ICC on smears depends on proper preparation and fixation. This review offers practical protocols and insights to help laboratories optimize ICC on cytological smears. Further research and standardization are necessary to enhance reproducibility and reliability of ICC on smears. The practical information provided is based on personal experience in our laboratory. When using cytological smears for Immunocytochemistry (ICC), it is essential to be aware of the importance of pre‐analytics to obtain confident results. The validation of antibodies on non‐CB preparations is an essential step for ensuring the accuracy of laboratory results. ICC is an essential ancillary technique that can be performed on cytological smears and its success relies on accurate specimen collection, handling, appropriate triage and processing. Detailing preparation, fixation and protocols for nuclear and cytoplasmic antibodies represents the focus of this review.