This article demonstrates a tour-de-force of this technology for the analysis of human hematologic cell types. The differentiation states of hematopoietic cells can be determined by "cluster of differentiation" (CD) markers. To create an "immunophenotype" panel, antibodies that monitored 13 core CD surface markers and 18 subset-specific cell-CD surface markers were used. In addition, a second panel was constructed using antibodies containing the same 13 core markers as well as 18 intracellular markers reflective of intracellular signaling events (e.g., through phosphoprotein detection). In addition three labels for total DNA, cell length, and cell viability were also used, so each panel measured 34 parameters.
Comparing the data for various phosphoproteins from atomic mass cytometry with conventional fluorescence cyotometry showed that both qualitatively and quantitatively similar patterns were obtained with each method. The data can be exported as typical .fcs files but the wealth of data generated is overwhelming. To address this issue, data analysis was performed using SPADE (spanning-tree progression analysis of densitynormalized events; see: Qiu et al., Nat Biotechnol 2011;29:886–891). SPADE analysis provides unsupervised clustering of the data and the results can be visualized with two-dimensional tree plots.
For example, in the immunophenotype panel, each node in the tree plot represents a cell cluster having a similar phenotype in the 13 dimensional space defined by the core surface markers. Well-defined cell types such as T cells or monocytes can provide internal standards for the nodes that enabled capturing unexpected transitional cell types and defining lineage by determining the relationships between each cell cluster. The functional state of each cell type can be similarly treated and visualized. Significant signaling events within each subtype were examined.
In addition to providing a system-level view of hematopoiesis this study examines pharmacological modulation with well-known kinase inhibitors such as dasatinib, which points to polypharmacology, which may explain the efficacy of this compound in certain B-cell malignancies. This approach demonstrates a method for the analysis of pathways using the resolving power of atomic mass spectrometry. Assay development of multiplexed formats using fluorophores is difficult due to the large differences in fluorescent brightness between probes and the spectral overlap. In mass cytometry the isotope signals vary within a twofold range and the isotopic masses are readily separated upon detection, which allows for a much greater number of parameters to be simultaneously measured and more facile assay optimization. The technology demonstrates a unique approach to high content methods that enables pathway analysis from primary cell types.