Evaluating immunocytochemistry performance in lung cancer cytology: An IASLC Cytology Working Group study

BACKGROUND: Immunohistochemistry (IHC) is essential for tumor subtyping and predictive biomarker analysis in non-small cell lung carcinoma (NSCLC). When only cytological material is available and no concurrent biopsy or cell block exists, immunocytochemistry (ICC) may need to be performed on slides prepared using diverse fixation protocols, introducing substantial pre-analytical variability. METHODS: Seven laboratories representing commonly used cytology fixation protocols were selected based on an exploratory global survey of 63 institutions. Two central laboratories prepared direct smears, cytospins and provided cells in PreservCyt from specimens with confirmed marker expression using the selected fixation protocols. Materials were distributed for immunocytochemical demonstration of TTF-1, p40, ALK, and ROS1 according to local protocols. Digitized slides were independently evaluated using standardized four-tier scoring criteria. RESULT: A total of 105 stained slides from 17 study runs were assessed. Delaunay-fixed, Papanicolaou-stained, and methanol-fixed polyethylene glycol-coated preparations demonstrated consistently superior ICC performance compared with other evaluated fixation protocols. In contrast, high rates of suboptimal staining were observed with PreservCyt fixation (81.8%) and air-dried preparations (58.8%). CONCLUSIONS: This study demonstrates that fixation protocol significantly impacts ICC performance on non-cell block cytological preparations. The observed fixation-dependent differences underscore the need for harmonized pre-analytical practices, robust local validation, and external quality assessment.