Immunophenotyping and Intracellular Staining of Fixed Whole Blood for Mass Cytometry (CyTOF)

Background

Non-invasive methods to obtain biological samples and single cell technologies are much sought after, as they provide an opportunity to comprehensively study human diseases. Blood not only provides for a minimally-invasive, cost effective and readily accessible source of immunological sample, but whole blood stimulation also serves as the closest mimic of the in vivo condition. Single-cell Mass Cytometry (or Cytometry by Time of Flight mass spectrometry, CyTOF) is ideally suited to broad profiling of the immune system, since it allows for > 40 parameter panels, with little to no spillover between channels, which is a significant advancement over the procedural limitations of fluorescence flow cytometry (Leipold et al., 2015). Conversely, cell acquisition speed is significantly lower, and cell loss significantly higher, for CyTOF compared to fluorescence flow cytometry. In this report, we show the establishment of a reference panel of 39-anti human antibodies for mass cytometry that broadly identifies the major immune cell types, well established T and B cell subpopulations, activation markers, cytolytic markers and cytokines. To characterize these immune cell lineages and their functional states, the panel of anti-human heavy metal-conjugated monoclonal antibodies was selected to target the epitopes shown in Table 3, and to be compatible with fixed whole blood samples (Fernandez and Maecker, 2015 and our unpublished data). The panel described here, comprises an almost equal number of pre-made and in-house conjugates. To build this panel, we implemented the recommended panel design guidelines for mass cytometry. Low-abundance targets were allocated to higher-sensitivity channels and antibodies were designated to channels to minimize potential spectral overlap (Takahashi et al., 2017). All antibodies in the designed panel were then titrated to best discriminate the positive population from the negative. This panel can be further customized for specific hypothesis driven studies. In addition, by integrating barcoding (Behbehani et al., 2014) within this framework, we also demonstrate the compatibility of this panel with large clinical studies, to minimize technical variability. The ability to freeze fixed blood samples is also convenient for assembly of sample sets for retrospective batched analysis, with decreased biosafety risks in the setting of infectious disease. With regard to SARS-CoV-2 or other infectious agents that might be present, whole blood fixation by the method described here has not been proven to be fully inactivating. But given the known effects of fixation on viral infectivity (Möller et al., 2015), it provides an increased level of safety.