Orion™: 15-plex single-step stain and imaging of tonsil tissue with reactive lymphoid hyperplasia

Immune cell function depends on where the cell sits in the tissue, so phenotype has to be read in place; this SITC 2023 poster stains a whole-slide tonsil section with reactive lymphoid hyperplasia using a 15-plex immuno-oncology panel in one staining round and images it in a single Orion scan.

Presented by RareCyte.

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• 15 markers, one staining round, one scan — then H&E on the same section. The FFPE tonsil section was stained in a single staining process with a 15-plex immuno-oncology biomarker panel, using ArgoFluor-conjugated antibodies and standard IF protocols, and imaged whole-slide at 20X on the Orion instrument in one imaging round, with tissue autofluorescence isolated as an additional channel and OME-TIFF written for viewing and quantification; the same section was then de-coverslipped and H&E-stained, so biomarker phenotype and classical morphology come from one piece of tissue.
• The panel resolves tonsil architecture compartment by compartment. Ki67 and PCNA co-expression marks the proliferative germinal center, Hoechst the densely packed mantle zone surrounding it, CD4 the interfollicular T-cell zones and CD20 the B-cell follicles, while Pan-Cytokeratin and E-Cadherin outline the epithelial crypts; CD45RO with CD4 identifies memory helper T cells, and CD31 marks the high endothelial venules through which CD4, FoxP3, CD8 and CD20 lymphocytes re-enter the tissue.
• PD-L1 registers at 3 distinct levels across one section, and PD-1 is restricted to germinal-center CD4 T cells in this field. PD-L1 is high in the tonsillar crypt epithelium, moderate in follicle germinal centers and low in stromal cells of the T-cell zone; PD-1 marks CD4 helper T cells inside the germinal center but not those in the T-cell zone and not CD8 cytotoxic T cells, while germinal-center macrophages express CD68 alone and macrophages outside the follicle co-express CD163 and CD68.