[show abstract][hide abstract] ABSTRACT: Mass cytometry addresses the analytical challenges of polychromatic flow cytometry by using metal atoms as tags rather than fluorophores and atomic mass spectrometry as the detector rather than photon optics. The many available enriched stable isotopes of the transition elements can provide up to 100 distinguishable reporting tags, which can be measured simultaneously because of the essential independence of detection provided by the mass spectrometer. We discuss the adaptation of traditional inductively coupled plasma mass spectrometry to cytometry applications. We focus on the generation of cytometry-compatible data and on approaches to unsupervised multivariate clustering analysis. Finally, we provide a high-level review of some recent benchmark reports that highlight the potential for massively multi-parameter mass cytometry.
Cancer Immunology and Immunotherapy 04/2013; · 3.64 Impact Factor
[show abstract][hide abstract] ABSTRACT: To transform the linear fluorescence intensity scale obtained with fluorescent microspheres to an antibody bound per cell (ABC) scale, a biological cell reference material is needed. Optimally, this material should have a reproducible and tight ABC value for the expression of a known clinical reference biomarker. In this study, we characterized commercially available cryopreserved peripheral blood mononuclear cells (PBMCs) and two lyophilized PBMC preparations, Cyto-Trol and PBMC-National Institute for Biological Standard and Control (NIBSC) relative to freshly prepared PBMC and whole blood samples. It was found that the ABC values for CD4 expression on cryopreserved PBMC were consistent with those of freshly obtained PBMC and whole blood samples. By comparison, the ABC value for CD4 expression on Cyto-Trol is lower and the value on PBMC-NIBSC is much lower than those of freshly prepared cell samples using both conventional flow cytometry and CyTOF™ mass cytometry. By performing simultaneous surface and intracellular staining measurements on these two cell samples, we found that both cell membranes are mostly intact. Moreover, CD4(+) cell diameters from both lyophilized cell preparations are smaller than those of PBMC and whole blood. This could result in steric interference in antibody binding to the lyophilized cells. Further investigation of the fixation effect on the detected CD4 expression suggests that the very low ABC value obtained for CD4(+) cells from lyophilized PBMC-NIBSC is largely due to paraformaldehyde fixation; this significantly decreases available antibody binding sites. This study provides confirmation that the results obtained from the newly developed mass cytometry are directly comparable to the results from conventional flow cytometry when both methods are standardized using the same ABC approach.
Cytometry Part A 04/2012; 81(7):567-75. · 3.71 Impact Factor
[show abstract][hide abstract] ABSTRACT: Flow cytometry is an essential tool for dissecting the functional complexity of hematopoiesis. We used single-cell "mass cytometry" to examine healthy human bone marrow, measuring 34 parameters simultaneously in single cells (binding of 31 antibodies, viability, DNA content, and relative cell size). The signaling behavior of cell subsets spanning a defined hematopoietic hierarchy was monitored with 18 simultaneous markers of functional signaling states perturbed by a set of ex vivo stimuli and inhibitors. The data set allowed for an algorithmically driven assembly of related cell types defined by surface antigen expression, providing a superimposable map of cell signaling responses in combination with drug inhibition. Visualized in this manner, the analysis revealed previously unappreciated instances of both precise signaling responses that were bounded within conventionally defined cell subsets and more continuous phosphorylation responses that crossed cell population boundaries in unexpected manners yet tracked closely with cellular phenotype. Collectively, such single-cell analyses provide system-wide views of immune signaling in healthy human hematopoiesis, against which drug action and disease can be compared for mechanistic studies and pharmacologic intervention.
[show abstract][hide abstract] ABSTRACT: The ion chemistry is discussed for fuel-rich, nearly sooting, methane–oxygen flames at atmospheric pressure with added acetylene. Different types of ion–molecule reactions, both positive and negative, which can contribute through chemical ionization (CI) processes are summarized including their dependence on temperature, pressure, and equivalence ratio . Extensive data were presented previously involving ion concentration profiles measured with a mass spectrometer as a function of distance along the axis of conical flames. An understanding of the dominant CI processes provides insight into the early chemical stage of soot formation associated with the flame reaction zone. The negative ion profiles show moderately unsaturated hydrocarbon ions upstream formed by proton transfer followed by progressive dehydrogenation; the highly unsaturated, carbonaceous ions observed downstream appear to arise by two- and three-body electron attachment, charge transfer, and H-atom stripping. The negative hydrocarbon ions can all be explained in terms of polyacetylene derivatives. The same build-up of carbonaceous species downstream is evident from the positive ion profiles. A major role is ascribed to proton transfer reactions with lesser contributions from charge transfer and ion–molecule condensation; three-body association is probably insignificant. Experiments with added acetylene indicate extensive fuel pyrolysis early in the reaction zone. There is no evidence that an ionic mechanism is dominant in forming soot precursors compared with neutral condensation reactions. Because of complexities in the positive ion chemistry, the negative ions appear to provide the more straightforward probe of the underlying neutral chemistry.
Canadian Journal of Chemistry 02/2011; 60(22):2766-2776. · 0.96 Impact Factor
[show abstract][hide abstract] ABSTRACT: Rate constants measured with the flowing afterglow technique at 298 ± 2 K are reported for the proton-transfer reactions of H3O+ with CH2O, CH3CHO, (CH3)2CO, HCOOH, CH3COOH, HCOOCH3, CH3OH, C2H5OH, (CH3)2O, and CH2CO. Dissociative proton-transfer was observed only with CH3COOH. The rate constants are compared with the predictions of various theories for ion–molecule collisions. The protonation is discussed in terms of the energetics and mechanisms of various modes of dissociation.
Canadian Journal of Chemistry 02/2011; 57(12):1518-1523. · 0.96 Impact Factor
[show abstract][hide abstract] ABSTRACT: Flowing afterglow measurements are reported which provide rate constants and product identifications at 298 ± 2 K for the gas-phase reactions of OH− with CH3OH, C2H5OH, CH3OCH3, CH2O, CH3CHO, CH3COCH3, CH2CO, HCOOH, HCOOCH3, CH2=C=CH2, CH3—C≡CH, and C6H5CH3. The main channels observed were proton transfer and solvation of the OH−. Hydration with one molecule of H2O was observed either to reduce the rate slightly and lead to products which are the hydrated analogues of the "nude" reaction, or to stop the reaction completely, k ≤ 10−12 cm3 molecule−1 s−1. The reaction of OH−•H2O with CH3—C≡CH showed an uncertain intermediate behaviour.
Canadian Journal of Chemistry 02/2011; 59(11):1615-1621. · 0.96 Impact Factor
[show abstract][hide abstract] ABSTRACT: The natural hydrocarbon ions CnHx± (n ≥ 2, x ≥ 0) present in premixed, fuel-rich, nearly sooting, CH4–C2H2–O2 flames at atmospheric pressure were studied as a probe of the early chemical stages of soot formation. Ion concentration profiles were measured mass-spectrometrically along the flame axis through the reaction zone into the burnt gas downstream. Total ionization profiles were examined for their dependence on both temperature and equivalence ratio, Families of individual CnHx− negative ion profiles exhibit concentration peaks in three distinct regions; predominantly oxygenated ions occur upstream, giving way to moderately unsaturated hydrocarbon ions near the end of the reaction zone, leading to highly unsaturated carbonaceous ions further downstream. The concentrations of the downstream ions alternate with the parity of n, the even-n species being larger. Series of CnHx+ positive ion profiles, for a given n, show profile peak positions which move steadily downstream with decreasing x, indicative of progressive dehydrogenation. The positive ion chemistry of these series is essentially independent of n. As is increased at constant temperature towards the sooting point, the concentrations of CnHx± ions increase while those of the oxygenated ions decrease; the positive ions show a relative enhancement of species having high values of n.
Canadian Journal of Chemistry 02/2011; 59(12):1760-1770. · 0.96 Impact Factor
[show abstract][hide abstract] ABSTRACT: Rate constants measured with the flowing afterglow technique at 297 ± 2 K are reported for the protonation of CH2O by H3+, N2H+, CH5+, HCO+, C2H5+, H3O+, H3S+, and HCNH+ and for the subsequent deprotonation by NH3. The rate constants are compared with predictions of various theories for ion–molecule collisions. The protonation was observed to proceed in the absence of competing channels and further decomposition and is discussed in terms of the energetics of the two sites of protonation and the energetics and mechanism of H2 elimination. The rate measurements provide evidence for the room-temperature conversion of the adduct C2H3+•H2 to the more stable isomer derived from the direct protonation of C2H4.
Canadian Journal of Chemistry 02/2011; 57(18):2350-2354. · 0.96 Impact Factor
[show abstract][hide abstract] ABSTRACT: This review paper describes a new technology, mass cytometry, that addresses applications typically run by flow cytometer analyzers, but extends the capability to highly multiparametric analysis. The detection technology is based on atomic mass spectrometry. It offers quantitation, specificity and dynamic range of mass spectrometry in a format that is familiar to flow cytometry practitioners. The mass cytometer does not require compensation, allowing the application of statistical techniques; this has been impossible given the constraints of fluorescence noise with traditional cytometry instruments. Instead of "colors" the mass cytometer "reads" the stable isotope tags attached to antibodies using metal-chelating labeling reagents. Because there are many available stable isotopes, and the mass spectrometer provides exquisite resolution between detection channels, many parameters can be measured as easily as one. For example, in a single tube the technique allows for the ready detection and characterization of the major cell subsets in blood or bone marrow. Here we describe mass cytometric immunophenotyping of human leukemia cell lines and leukemia patient samples, differential cell analysis of normal peripheral and umbilical cord blood; intracellular protein identification and metal-encoded bead arrays.
Journal of immunological methods 09/2010; 361(1-2):1-20. · 2.35 Impact Factor
[show abstract][hide abstract] ABSTRACT: We examine the suitability of metal-containing polystyrene beads for the calibration of a mass cytometer instrument, a single particle analyser based on an inductively coupled plasma ion source and a time of flight mass spectrometer. These metal-containing beads are also verified for their use as internal standards for this instrument. These beads were synthesized by multiple-stage dispersion polymerization with acrylic acid as a comonomer. Acrylic acid acts as a ligand to anchor the metal ions within the interior of the beads. Mass cytometry enabled the bead-by-bead measurement of the metal-content and determination of the metal-content distribution. Beads synthesized by dispersion polymerization that involved three stages were shown to have narrower bead-to-bead variation in their lanthanide content than beads synthesized by 2-stage dispersion polymerization. The beads exhibited insignificant release of their lanthanide content to aqueous solutions of different pHs over a period of six months. When mixed with KG1a or U937 cell lines, metal-containing polymer beads were shown not to affect the mass cytometry response to the metal content of element-tagged antibodies specifically attached to these cells.
[show abstract][hide abstract] ABSTRACT: 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 (<3% cerium oxide ratio). At mass resolution (full width at half-maximum) M/DeltaM > 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 <15 elemental tags are used, a higher sensitivity mode at lower resolution (M/DeltaM > 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.
[show abstract][hide abstract] ABSTRACT: We have investigated the possibility of using element-tagged antibodies for protein detection and quantification in microplate format using Inductively Coupled Plasma Mass Spectrometry (ICP-MS), and compared the results to conventional immunoassays, such as Enzyme-Linked Immunosorbent Assay (ELISA) and Western blotting. The technique was further employed to detect low levels and measure DNA-binding activity of transcription factor p53 in leukemia cell lysates through its interaction with immobilized oligonucleotides and recognition by element-tagged antibodies. The advantages of ICP-MS detection for routine performance of immunoassays include increased sensitivity, wide dynamic range, minimal interference from complex matrices, and high throughput. Our approach advances the ICP-MS technology and demonstrates its applicability to proteomic studies through the use of antibodies directly labeled with polymer tags bearing multiple atoms of lanthanides. Development of this novel methodology will enable fast and quantitative identification of multiple analytes in a single well.
Journal of Immunological Methods 08/2008; 336(1):56-63. · 2.23 Impact Factor
[show abstract][hide abstract] ABSTRACT: The enumeration of absolute cell numbers and cell proliferation in clinical samples is important for diagnostic and research purposes. Detection of cellular DNA with fluorescent dyes is the most commonly used approach for cell enumeration in cytometry. Inductively coupled plasma mass spectrometry (ICPMS) has been recently introduced to the field of protein and cell surface antigen identification via ICPMS-linked immunoassays using element-labeled affinity reagents such as gold and lanthanide-conjugated antibodies. In the present work, we describe novel methods for using metallointercalators that irreversibly bind DNA and low concentrations of rare earth metals added to cell growth media for rapid and sensitive measurement of cell numbers by mass spectrometry. We show that Ir- and Rh-containing metallointercalators are useful reagents for labeling cells and normalizing signals when studying antigen expression on different types and numbers of cells. Results are presented for solution analysis performed by conventional ICPMS and compared to measurements obtained on the novel flow cytometer mass spectrometer (FC-MS) instrument, designed to analyze multiple antigens and DNA simultaneously in single cells.
[show abstract][hide abstract] ABSTRACT: Advances in the development of highly multiplexed bio-analytical assays with inductively coupled plasma mass spectrometry (ICP-MS) detection are discussed. Use of novel reagents specifically designed for immunological methods utilizing elemental analysis is presented. The major steps of method development, including selection of elements for tags, validation of tagged reagents, and examples of multiplexed assays, are considered in detail. The paper further describes experimental protocols for elemental tagging of antibodies, immunostaining of live and fixed human leukemia cells, and preparation of samples for ICP-MS analysis. Quantitative analysis of surface antigens on model cell lines using a cocktail of seven lanthanide labeled antibodies demonstrated high specificity and concordance with conventional immunophenotyping.
[show abstract][hide abstract] ABSTRACT: Recent progress in the development of massively multiplexed bioanalytical assays using element tags with inductively coupled plasma mass spectrometry detection is reviewed. Feasibility results using commercially available secondary immunolabeling reagents for leukemic cell lines are presented. Multiplex analysis of higher order is shown with first generation tag reagents based on functionalized carriers that bind lanthanide ions. DNA quantification using metallointercalation allows for cell enumeration or mitotic state differentiation. In situ hybridization permits the determination of cellular RNA. The results provide a feasibility basis for the development of a multivariate assay tool for individual cell analysis based on inductively coupled plasma mass spectrometry in a cytometer configuration.
Spectrochimica Acta Part B Atomic Spectroscopy 01/2007; 62:188-195. · 3.14 Impact Factor
[show abstract][hide abstract] ABSTRACT: A novel electrospray ion source/selected ion flow tube/triple quadrupole detector mass spectrometer has been constructed for the study of the kinetics and product distributions of reactions of a diverse array of electrosprayed ions reacting with neutral molecules. The electrospray source provides a near-universal source for ions of biological and other interests. The operation of the instrument is demonstrated with a selection of reactivity and kinetic measurements and threshold collision-induced dissociation determinations. Reactions investigated include H/D exchange between protonated bradykinin and deuterated ammonia, the protonation of the oligo-nucleotide anion [AGTCTG − 5H+]5− with HBr, the ozonolysis of Ca2+, the reaction of protonated phthalic anhydride with dimethyl disulfide, and the Argon collision-induced dissociation of Ba(C6H6)2+.
International Journal of Mass Spectrometry. 01/2007;
[show abstract][hide abstract] ABSTRACT: There is a great need in cell biology for the simultaneous detection of many intracellular and extracellular proteins within single cells. Current optical methods based on fluorescence activated flow cytometry are difficult to multiplex. We have developed a novel application of ICP-MS-linked metal-tagged immunophenotyping which has great potential for highly multiplexed proteomic analysis. Expression of intracellular oncogenic kinase BCR/Abl, myeloid cell surface antigen CD33, human stem cell factor receptor c-Kit and integrin receptor VLA-4 were investigated using model human leukemia cell lines. Antigens to which specific antibodies are available and are distinguishably tagged can be determined simultaneously, or multiplexed. Four commercially available tags (Au, Sm, Eu, and Tb) conjugated to secondary antibodies enable a 4-plex assay assuming that the primary antibodies are not cross-reactive. Results obtained by ICP-MS were compared with data from FACS. ICP-MS as an analytical detector possesses several advantages that enhance the performance of immunoassays, which are discussed in detail. Although multiplexing using metal-conjugated reagents is in a very early stage of research and feasibility studies, it is already apparent that more than four antigens could be accurately detected simultaneously using the ICP-MS instrument.
Journal of Immunological Methods 02/2006; 308(1-2):68-76. · 2.23 Impact Factor
[show abstract][hide abstract] ABSTRACT: Ion–molecule reactions that can be used for resolution of spectral overlaps of long-lived or stable (T1/2 > 100 years) atomic isobars on the long-lived radio-isotopes (100 < T1/2 < 1012 years) in mass spectrometry are considered. Results for the separations of isobaric overlaps via cation reactions with NO, N2O, O2, CO2, C2H2, CH3F studied with the Inductively Coupled Plasma Dynamic Reaction Cell™ Mass Spectrometer (ICP-DRC™-MS) with use of stable isotopes are presented. Overview of potential and reported reactions for separation of 35 isobars is given. Potential for the following isobaric pairs separation is shown (reaction gas and the extent of separation achieved to date in parenthesis): 32Si+/32S+(NO, 5 × 104), 40K+/40Ar+(N2O, 1.9 × 103), 40K+/40Ca+(N2O, 50), 59Ni+/59Co+(N2O, 6), 79Se+/79Br+(O2, 7.2 × 103), 81Kr+/81Br+(C2H2, 1.5 × 104), 93Mo+/93Nb+(N2O, 100), 93Mo+/93Zr+(N2O, 150), 135Cs+/135Ba+(N2O, 8 × 104), 137,138La+/137,138Ba+(O2, 40), 146Sm+/146Nd+(CO2, 1.5 × 105), 176Lu+/176Hf+(NO, 2.8 × 103), 187Re+/187Os+(N2O, 2.8 × 104). Effect of instrumental parameters on reactivity is discussed. The relevance of this work to accelerator mass spectrometry is discussed briefly.
International Journal of Mass Spectrometry. 01/2006;
[show abstract][hide abstract] ABSTRACT: Conventional gene expression profiling relies on using fluorescent detection of hybridized probes. Physical characteristics of fluorophores impose limitations on achieving a highly multiplex gene analysis of single cells. Our work demonstrates the feasibility of using metal-tagged in situ hybridization for mRNA detection by inductively coupled plasma mass spectrometry (ICP-MS). ICP-MS as an analytical detector has a number of unique and relevant properties: 1) metals and their stable isotopes generate non-overlapping distinct signals that can be detected simultaneously; 2) these signals can be measured over a wide dynamic range; 3) ICP-MS is quantitative and very sensitive. We used commercial antibodies conjugated to europium (Eu) and gold together with biotinylated oligonucleotide probes reacted with terbium-labeled streptavidin to demonstrate simultaneous mRNA and protein detection by ICP-MS in leukemia cells.
[show abstract][hide abstract] ABSTRACT: The concept of the effective potential provides inadequate estimation of the ion energy in RF-driven quadrupole fields. It is especially limited for the quantitation of additional enthalpy introduced by the RF-driven field for an ion molecule reaction. On the contrary, the effective potential is clearly successful in describing of the ion oscillation frequency, and has been surprisingly powerful considering the strict limitations of the adiabatic approximation. This work is an attempt to understand this contradiction and to consider a novel concept of the estimation of the ion energy in an RF field. This theoretical model for the averaged ion energy is useful in describing specific features of the RF field contribution to the enthalpy of ion molecule reactions. To demonstrate this effect, several ion molecule reactions are investigated experimentally including cluster formation, endothermic and exothermic oxidation reactions.
International Journal of Mass Spectrometry - INT J MASS SPECTROM. 01/2005;