Sequential same slide multiplex immunofluorescence and H&E staining for combined phenotypic and morphologic characterization of formalin-fixed paraffin-embedded tissue sections

Multiplex immunofluorescence and H&E normally run on two different serial sections, which no longer contain the same cells and are not always obtainable when tissue is limited; this AACR 2020 poster stains and images both on a single slide, in sequence, on one instrument.

Presented by RareCyte, Ultivue, and Indica Labs.

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• Multiplex immunofluorescence and H&E ran in sequence on one physical slide, not on two serial sections. In FFPE samples from 3 serial sections of tonsil and primary colon and lung tumor biopsies, CD8, CD68, PD-L1 and pan-Cytokeratin were stained with Ultivue’s UltiMapper I/O PD-L1 kit on a Leica Biosystems BOND RX, imaged in five spectrally distinct fluorescence channels (DAPI, FITC, TRITC, Cy5, Cy7) on the RareCyte CyteFinder II HT, then de-coverslipped, stained with H&E and re-imaged in brightfield on that same instrument — so the morphology and the protein phenotypes describe the identical cells.
• A classifier trained on the H&E image separated tumor from stroma as well as one trained on the fluorescent cytokeratin and DAPI signal. Across the colon and lung sections, HALO AI segmentation gave per-region cell counts the authors describe as comparable, which is what leads them to conclude that a tumor-specific biomarker could be dropped from the fluorescence panel when the H&E carries the tissue architecture. Read that as their qualitative call rather than a measured equivalence: Figure 2 plots the counts on a log axis and reports no statistical test, and Lung 5 had to borrow Lung 6’s tumor/stroma segmentation after the tissue was damaged during de-coverslipping.
• Cell phenotypes were counted inside the tissue regions the H&E defined, not across the section as a whole. HALO Highplex FL scored CD8+ cytotoxic T-cells, CD68+ macrophages, CD68+/PD-L1+ immuno-suppressive macrophages, pan-CK+ tumor cells and pan-CK+/PD-L1+ immune-evading tumor cells within the tumor and stromal regions, and the authors report the UltiMapper I/O PD-L1 kit stained reproducibly across the serial sections. RareCyte instruments are for research use; H&E is a clinical stain, but this workflow makes no diagnostic claim.