Mass Cytometry for the Assessment of Immune Reconstitution After Hematopoietic Stem Cell Transplantation

A novel instrument for real time analysis of individual biological cells or other microparticles is described. The instrument is based on inductively coupled plasma time-of-flight mass spectrometry and comprises a three-aperture plasma-vacuum interface, a dc quadrupole turning optics for decoupling ions from neutral components, an rf quadrupole ion guide discriminating against low-mass dominant plasma ions, a point-to-parallel focusing dc quadrupole doublet, an orthogonal acceleration reflectron analyzer, a discrete dynode fast ion detector, and an 8-bit 1 GHz digitizer. A high spectrum generation frequency of 76.8 kHz provides capability for collecting multiple spectra from each particle-induced transient ion cloud, typically of 200-300 micros duration. It is shown that the transients can be resolved and characterized individually at a peak frequency of 1100 particles per second. Design considerations and optimization data are presented. The figures of merit of the instrument are measured under standard inductively coupled plasma (ICP) operating conditions ( 900 for m/z = 159, the sensitivity with a standard sample introduction system of >1.4 x 10(8) ion counts per second per mg L(-1) of Tb and an abundance sensitivity of (6 x 10(-4))-(1.4 x 10(-3)) (trailing and leading masses, respectively) are shown. The mass range (m/z = 125-215) and abundance sensitivity are sufficient for elemental immunoassay with up to 60 distinct available elemental tags. When 500) can be used, which provides >2.4 x 10(8) cps per mg L(-1) of Tb, at (1.5 x 10(-3))-(5.0 x 10(-3)) abundance sensitivity. The real-time simultaneous detection of multiple isotopes from individual 1.8 microm polystyrene beads labeled with lanthanides is shown. A real time single cell 20 antigen expression assay of model cell lines and leukemia patient samples immuno-labeled with lanthanide-tagged antibodies is presented.

Multiparametric single-cell analysis is critical for understanding cellular heterogeneity. Despite recent technological advances in single-cell measurements, methods for analyzing high-dimensional single-cell data are often subjective, labor intensive and require prior knowledge of the biological system under investigation. To objectively uncover cellular heterogeneity from single-cell measurements, we present a novel computational approach, Spanning-tree Progression Analysis of Density-normalized Events (SPADE). We applied SPADE to cytometry data of mouse and human bone marrow. In both cases, SPADE organized cells in a hierarchy of related phenotypes that partially recapitulated well-described patterns of hematopoiesis. In addition, SPADE produced a map of intracellular signal activation across the landscape of human hematopoietic development. SPADE revealed a functionally distinct cell population, natural killer (NK) cells, without using any NK-specific parameters. SPADE is a versatile method that facilitates the analysis of cellular heterogeneity, the identification of cell types, and comparison of functional markers in response to perturbations.