Introduction of a robust workflow for the whole-slide acquisition and co-registration of multiplex immunofluorescence tissue images for analysis of a 9-color, 8-marker immunophenotyping assay

Profiling multiple cell types in tumor tissue takes more markers than conventional immunohistochemistry can support; this AACR 2020 poster from Indica Labs, RareCyte and Ultivue stains 8 markers in a single run, images them across 2 rounds on a CyteFinder II HT scanner, and co-registers the rounds into one 10-channel whole-slide image for cell-level phenotyping.

Presented by Indica Labs / RareCyte / Ultivue.

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• Eight markers were stained in one run, then imaged in two rounds of four. The UltiMapper I/O Immuno8 kit stains all 8 antibodies together on a Leica Bond automated stainer and amplifies every barcode in the same run; imaging probes complementary to one set of 4 barcodes are then hybridized and scanned on the RareCyte CyteFinder II HT, dehybridized, and replaced with probes for antibodies 5 to 8 for a second scan, building the 9-color panel of CK, CD3, CD4, FoxP3, CD8a, PD-1, PD-L1 and CD68 alongside DAPI.
• The two imaging rounds were co-registered at the cellular level and fused into a single 10-channel image. The ome.tiff files from rounds 1 and 2 were aligned with the Serial Stain Registration tool in HALO 3.1, then fused by the Serial Stain/Section module into one 10-channel slide; overlaying each round’s DAPI channel rendered well-aligned nuclei white and misaligned nuclei blue or yellow, and those misaligned nuclei were seen mainly in areas of tissue disturbed between rounds — necrotic areas where cellular debris rinsed off, and small movements or folds in the tissue.
• Nuclear detection was keyed to the second round’s DAPI, and one sample was excluded from the results. The kit stained FFPE colorectal cancer, non-small cell lung cancer and human tonsil control tissue; the HighPlex FL module then segmented individual cells and analyzed nuclear, cytoplasm or membrane biomarkers, with nuclear detection based on the round 2 DAPI signal so that only cells that made it through both rounds of staining were quantified, and Sample Lung 3 was excluded from the results for low antigen retrieval during automated staining. Phenotypes were resolved by marker combination, including cytotoxic T cells (CD3+ and CD8+), regulatory T cells (CD3+, CD4+ and FoxP3+) and macrophages (CD68+).