Implementing Mass Cytometry at the Bedside to Study the Immunological Basis of Human Diseases: Distinctive Immune Features in Patients with a History of Term or Preterm Birth

Abstract

Single-cell technologies have immense potential to shed light on molecular and biological processes that drive human diseases. Mass cytometry (or Cytometry by Time Of Flight mass spectrometry, CyTOF) has already been employed in clinical studies to comprehensively survey patients’ circulating immune system. As interest in the “bedside” application of mass cytometry is growing, the delineation of relevant methodological issues is called for. This report uses a newly generated dataset to discuss important methodological considerations when mass cytometry is implemented in a clinical study. Specifically, the use of whole blood samples versus peripheral blood mononuclear cells (PBMCs), design of mass-tagged antibody panels, technical and analytical implications of sample barcoding, and application of traditional and unsupervised approaches to analyze high-dimensional mass cytometry datasets are discussed. A mass cytometry assay was implemented in a cross-sectional study of 19 women with a history of term or preterm birth to determine whether immune traits in peripheral blood differentiate the two groups in the absence of pregnancy. Twenty-seven phenotypic and 11 intracellular markers were simultaneously analyzed in whole blood samples stimulated with lipopolysaccharide (LPS at 0, 0.1, 1, 10, and 100 ng mL−1) to examine dose-dependent signaling responses within the toll-like receptor 4 (TLR4) pathway. Complementary analyses, grounded in traditional or unsupervised gating strategies of immune cell subsets, indicated that the prpS6 and pMAPKAPK2 responses in classical monocytes are accentuated in women with a history of preterm birth (FDR<1%). The results suggest that women predisposed to preterm birth may be prone to mount an exacerbated TLR4 response during the course of pregnancy. This important hypothesis-generating finding points to the power of single-cell mass cytometry to detect biologically important differences in a relatively small patient cohort.

Mapping the Fetomaternal Peripheral Immune System at Term Pregnancy

“…To ensure comparability between acquired samples, all blood samples were processed using a standardized protocol for stimulation, fixation, storage and processing of whole blood samples for mass cytometry analysis (10–12). To examine the function of fetal and maternal immune cells, whole blood samples were stimulated with a control vehicle to assess endogenous cellular function (unstimulated condition), the Toll-like receptor 4 (TLR4) agonist LPS (LPS condition), or a combination of extracellular cytokines eliciting receptor-specific intracellular responses (interleukin (IL)-2, IL-6, granulocyte macrophage colony-stimulating factor (GM-CSF), and interferon (INF) α2A).…”

“…All antibodies conjugated in-house as well as those obtained pre-conjugated were titrated and validated on samples that were processed identically to the samples used in the study. Abs were tested for specificity and sensitivity across a range of concentrations (typically, 0.25 to 4 µg/mL) as described in Gaudilliere et al (10). All but one Ab (GATA3) produced consistent antigen-specific staining above background levels.…”

Optimization of Receptor Occupancy Assays in Mass Cytometry: Standardization Across Channels with QSC Beads

“…RO assays generally involve measuring bound drug relative to total target receptor on single cells by flow cytometry. Mass cytometry has rapidly evolved to become a relevant tool in several fields of translational clinical research . In mass cytometry, antibodies are conjugated to purified metal isotopes instead of fluorophores, which dramatically reduces signal overlap and allows simultaneous detection of more than 40 parameters in individual cells by inductively‐coupled plasma mass spectrometry .…”

Atomic mass tag of bismuth‐209 for increasing the immunoassay multiplexing capacity of mass cytometry

“…This combination of these two technologies combines their strengths by offering single-cell assays with measurements of over 50 simultaneously detected parameters. This advance significantly improves the analytical capabilities of single-cell cytometry and enables the high-dimensional investigation of basic biological and clinical research (7–9). Additional benefits in CyTOF technology have resulted in minimal spectral overlap between detection channels, absence of auto-fluorescence background and of natural contaminants, as well as promisingly enhanced sensitivity (10–15)…”