Multiplex Cryoimmunostaining in Fine-Needle Aspiration Biopsies and Effusion Samples: Preliminary Results

Introduction: Immunocytochemistry (ICC) on cytospins and cell blocks is usually limited to a single marker per slide, often restricting diagnostic accuracy when sample cellularity is low. Multiplex cryoimmunostaining (mCryo), though not yet used in a routine cytopathology practice, may help overcome these limitations. Our study evaluated the applicability of mCryo compared with standard ICC on routinely prepared cytospins. Methods: Residual fine-needle aspiration biopsy (FNAB) and effusion samples from various diseases were used. mCryo staining with antibody panel consisting of EpCAM, CK7, CD56, TTF-1, p40, CD45 and calretinin (X-ZELL, Singapore) was tested on methanol-fixed cytospins, corresponding ICC-stained cytospins served as controls. All slides were reviewed blindly. Results: Forty-two samples were included in the analysis (21 carcinomas, eight lymphomas, five melanomas, eight non-neoplastic). mCryo revealed preserved morphology and no background staining in most of the cases. A 100% of true positivity for EpCAM (21/21), CK7 (21/21) p40 (3/3) and TTF-1 (1/1) in carcinomas matching ICC results. TTF-1 was also true positive in 2/2 benign thyroid nodule samples. CD45 was detected in all samples with both methods, labeling lymphocytes. Calretinin was true positive only in 2/6 effusion samples. CK7 positivity was unexpectedly detected in all melanomas (5/5) by mCryo but not by ICC. Moreover, CD56 was also positive in all carcinomas and melanomas by mCryo (26/26), while only in 9 cases by ICC, raising the question of unspecific staining, antibody cross-reactivity or spectral overlap of fluorochromes. Conclusions: mCryo enables simultaneous evaluation of multiple markers per slide on routinely prepared cytospins, offering potential diagnostic advantages for paucicellular samples. However, further method and panel improvement, and validation are essential for accurate diagnostic implementation into routine practice.