iCoreDrop: A robust immune monitoring spectral cytometry assay with six open channels for biomarker flexibility

Recent advances in spectral cytometry have extended our ability to monitor immune cell subsets and activation status while simultaneously improving rare population detection. However, technical challenges in reference control selection and autofluorescence extraction serve as barriers to broad application of spectral flow cytometry. Furthermore, the complexity of spectral cytometry panel development limits the adaptation of established assays. Here, we describe the development of a spectral immunophenotyping assay with robust drop-in capability to enable biomarker interrogation flexibility. The immune monitoring core (iCore), which can be used in part or total, captures broad and granular immune subsets across T cells, B cells, NK cells, monocytes, dendritic cells, and granulocytes in peripheral whole blood. Additional user-selected biomarkers can be dropped in (Drop) using channels BV421, Alexa Fluor 488, PE, PE-Cy7, APC, and APC-Cy7. A comprehensive assessment of reagent and panel performance was conducted, including reference control comparison and optimal autofluorescence (AF) extraction on the 5-laser Cytek Aurora system for healthy donor blood. Assay precision and stability analyses revealed robust intra-assay precision, with 95% of 83 distinct population gates having <20% CV. In the presence of additional drop-in markers in two different settings, a T cell module and a myeloid/B cell module, the drop-in channels themselves achieved <20% CV across 12 out of 13 additionally queried population gates. Overall, establishment of optimal unmixing practices will enable widespread adoption of spectral cytometry assays.