Abstract
Cytometry by Time-Of-Flight (CyTOF) uses antibodies conjugated to isotopically pure metals to identify and quantify a large number of cellular features with single-cell resolution. A barcoding approach allows for 20 unique samples to be pooled and processed together in one tube, reducing the intra-barcode technical variability. However, with only 20 samples per barcode, multiple barcode sets (batches) are required to address questions in robustly powered study designs. A batch adjustment procedure is required to reduce variability across batches and to facilitate direct comparison of runs performed across multiple barcodes run over weeks, months, or years. We describe a method using technical replicates that are included in each run to determine and apply an appropriate adjustment per batch without manual intervention. The use of technical replicate samples (i.e., anchors or reference samples) avoids assumptions of sample homogeneity among batches, and allows direct estimation of batch effects and appropriate adjustment parameters applicable to all samples within a batch. Quantification of cell subpopulations and mean signal intensity pre- and post-adjustment using both manual gating and unsupervised clustering demonstrate substantial mitigation of batch effects in the anchor samples used for this adjustment calculation, and in a second validation set of technical replicates.
Highlights
Mass cytometry, or Cytometry by Time-Of-Flight (CyTOF), is a high-throughput single-cell analysis technology that allows simultaneous measurement of 40+ cellular parameters via detection of rare earth heavy metal isotopes conjugated to monoclonal antibodies
The differences in the numbers of zero-valued events are troublesome for quantile normalization, which either inflates the zeros to non-zero values introducing noise, or squashes the lowest non-zero events to zero, resulting in loss of information
Quantile normalization can enforce identical distributions among replicates and produces the most consistent results on the single-channel Kolmogorov–Smirnov test (Figure 4A), we excluded this option from further analysis of cell population fraction variability due to the artifacts it introduces (Figure 4B)
Summary
Cytometry by Time-Of-Flight (CyTOF), is a high-throughput single-cell analysis technology that allows simultaneous measurement of 40+ cellular parameters via detection of rare earth heavy metal isotopes conjugated to monoclonal antibodies. The high-dimensionality of mass cytometry casts a wide exploratory net to allow: [1] discovery of novel cell phenotypes, [2] quantification of differences in cell-type composition (population frequency), and [3] analysis of marker expression levels (mean signal intensity) that may reflect different activation and/or functional cellular attributes. This systems immunology approach, when applied with relevant computational strategies, has the potential to unravel the cellular diversity and heterogeneity that underlies human immune-mediated disorders, providing descriptive and mechanistic insight with translational impact. Mass cytometry has been applied to study T cell proliferation and differentiation [5], macrophage phagocytosis [6], parallel DNA, RNA, and protein biosynthesis [7], and cell cycle status in malignancy and immunotherapy [8,9,10], to name a few applications
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