An Automated, Ventana Discovery Platform-based Imaging Workflow for Simultaneous Quantification of B Cells, Plasma...

Accurate, sensitive quantification of B-lineage cells is critical for pharmacodynamic evaluation of B cell-targeted therapies in lupus nephritis (LN) clinical trials. While high-dimensional discovery platforms offer broad profiling, they often lack the sensitivity, quantitative rigor, and throughput needed for precise cell enumeration in renal trial needle biopsies. Traditional immunostaining is hampered by CD20-directed therapeutic masking or downregulation, inadequate sensitivity of CD19 in FFPE tissue, and confounding renal tubular CD138 expression. This protocol details an automated, fit-for-purpose, 5-plex sequential tyramide signal amplification (TSA)-based immunofluorescence assay (CD38, CD79a, CD19, Ki-67, CD138) developed on the Ventana Discovery Ultra platform for deployment on single tissue sections. The workflow anchors B-cell detection on CD79a to ensure sensitivity and utilizes CD38 as an obligate co-marker for CD138+ antibody-secreting cells (ASCs) to definitively exclude the CD138+ epithelial background. Following acquisition via fluorescence whole-slide imaging, a digital analysis pipeline utilizing InstanSeg-based automated segmentation rigorously classifies cell phenotypes to generate precise spatial densities (cells/mm²). This validated protocol maximizes data yield from scarce clinical biopsies while providing high-precision quantitative monitoring of longitudinal therapeutic depletion in the renal microenvironment. Key features • Automated trial scalability: Details an automated, TSA-based 5-plex assay optimized for the Ventana Discovery Ultra platform, ensuring high-throughput, reproducible B-lineage quantification in multicenter cohorts. • Robust lineage identification: Establishes CD79a as an anchor for sensitive B-cell detection in FFPE tissue, avoiding CD20 therapeutic masking/downregulation and CD19 epitope instability. • High-specificity ASC enumeration: Resolves confounding CD138+ renal tubular background using an obligate CD38/CD138 co-labeling strategy to definitively identify ASCs within the renal microenvironment. • Single-section phenotypic resolution: Combines multiplexing and InstanSeg segmentation on one 4-μm section to simultaneously quantify B-lineage subsets in tissue-limited renal needle biopsies.