Spectral Flow Cytometry Methods and Pipelines for Comprehensive Immunoprofiling of Human Peripheral Blood and Bone Marrow.

Profiling hematopoietic and immune cells provides important information about disease risk, disease status, and therapeutic responses. Spectral flow cytometry enables high-dimensional single-cell evaluation of large cohorts in a high-throughput manner. Here, we designed, optimized, and implemented new methods for deep immunophenotyping of human peripheral blood and bone marrow by spectral flow cytometry. Two blood antibody panels capture 48 cell-surface markers to assess more than 58 cell phenotypes, including subsets of T cells, B cells, monocytes, NK cells, and dendritic cells, and their respective markers of exhaustion, activation, and differentiation in less than 2 ml of blood. A bone marrow antibody panel captures 32 markers for 35 cell phenotypes, including stem/progenitor populations, T cell subsets, dendritic cells, NK cells, and myeloid cells in a single tube. We adapted and developed innovative flow cytometric analysis algorithms, originally developed for single-cell genomics, to improve data integration and visualization. We also highlight technical considerations for users to ensure data fidelity. Our protocol and analysis pipeline accurately identifies rare cell types, discerns differences in cell abundance and phenotype across donors, and shows concordant immune landscape trends in patients with known hematologic malignancy.