Upping the "Plex": Whole Slide, Automated 14-Color Multiplex Immunofluorescence Staining of the Tumor...

Multiplex immunofluorescence (mIF) enables simultaneous visualization of multiple markers on a single slide. High-plex assays (>30 markers) typically rely on oligonucleotide (oligo)-tagged antibodies. Throughput of high-plex, oligo-based assays is limited, and results are not always comparable to gold-standard chromogenic immunohistochemistry (IHC), due to lack of amplification, particularly for immune checkpoint molecules such as PD-1 and PD-L1. In contrast, tyramide signal amplification (TSA)-based mIF improves detection sensitivity and throughput but is limited to fewer markers (5-8 markers). This study aimed to develop a combined oligo- and TSA-based mIF strategy to simultaneously detect 12-14 markers across whole-slide tissue sections, with equivalence to singleplex chromogenic immunohistochemistry (IHC) for each individual marker.

Various antibody stripping methods were evaluated, leading to optimization of a protocol using 2-mercaptoethanol/SDS/EDTA with heat to fully remove oligo-tagged antibodies. The finalized assay consisted of two phases. The first phase employed oligo-mIF to detect markers including CD3, CD4, CD8, CD68, PD-1, PD-L1, FoxP3, and a panCK/Sox10 cocktail, requiring two rounds of staining and imaging. Following complete removal of antibodies and fluorophores, a second phase using TSA-mIF was performed to detect LAG-3, Eomesodermin, T-bet, PD-1, PD-L1, and CD163.

The optimized stripping method successfully removed oligo-tagged antibodies, enabling sequential staining. Most oligo-mIF markers demonstrated comparable performance to gold-standard chromogenic IHC. However, PD-1 and PD-L1 did not achieve equivalency without amplification and only matched IHC performance when detected using TSA.

A combined oligo- and TSA-based mIF assay was successfully developed, enabling detection of up to 12 markers with performance comparable to IHC. TSA remains necessary for detecting low-mid level PD-1 and PD-L1 expression. Future optimization will retain PD-1 and PD-L1 in the TSA phase and replace their oligo-based counterparts with non-amplification-dependent markers, expanding the assay to 14 markers and enhancing whole-slide characterization of the tumor microenvironment.