Fixation protocol determined immunocytochemistry performance on lung cancer cytology slides in an IASLC study

Seven laboratories chosen after a survey of 63 institutions

Immunohistochemistry on a biopsy or a cell block is the usual basis for TTF-1 and p40 subtyping and for ALK and ROS1 testing in NSCLC. When the only material available is cytological and there is neither a concurrent biopsy nor a cell block, the stain may have to be done on slides prepared with whatever fixation the laboratory uses.

The working group surveyed 63 institutions and then selected seven laboratories whose fixation protocols were in common use. Two central laboratories prepared direct smears and cytospins, and supplied cells in PreservCyt, from specimens already known to express the markers. Each participating laboratory then stained the material according to its own local protocol.

Delaunay-fixed Pap-stained and PEG-coated methanol preparations stained most reliably

All slides were digitised and evaluated independently against a standardised four-tier scale, and 105 slides from 17 study runs went into the comparison. The preparations that gave consistently superior ICC performance were Delaunay-fixed, Papanicolaou-stained, and methanol-fixed polyethylene glycol (PEG)-coated preparations.

Because every specimen had confirmed expression of the marker being tested, the design measures whether a protocol can show a marker that is present. It does not estimate how often each protocol would miss expression in an unselected series of patient samples.

Suboptimal staining in 81.8% of PreservCyt and 58.8% of air-dried preparations

PreservCyt, a methanol-based liquid medium used to collect cells for liquid-based cytology, gave suboptimal staining in 81.8% of preparations. Air-dried preparations, which are commonly made for rapid on-site evaluation, were suboptimal in 58.8%.

A call for harmonised fixation, local validation and external quality assessment

The authors conclude that the fixation-dependent differences point to a need for change in laboratory practice.

“This study demonstrates that fixation protocol significantly impacts ICC performance on non-cell block cytological preparations.”Jain et al., Cancer Cytopathology (2026)

They recommend harmonised pre-analytical practices, robust local validation of each protocol used for ICC, and participation in external quality assessment.