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Immune monitoring technology primer: flow and mass cytometry
© Maecker and Harari. 2015
- Received: 31 July 2015
- Accepted: 6 August 2015
- Published: 15 September 2015
- Light Scatter
- Dimension Reduction Technique
- Absolute Cell Count
- Mass Cytometry
- Flow Cytometry Standard
Flow and mass cytometry uniquely allow for the quantitation of multiple parameters on many individual cells. Up to 17 or more parameters are possible with fluorescence, while 40 or more parameters can be quantitated with mass cytometry . It is not unusual to collect data on 105–107 cells per sample by either technique. The data from each sample is compiled in a Flow Cytometry Standard (FCS) file, for both flow and mass cytometry. The FCS file lists the intensities obtained for each probe on each individual cell. Analysis of FCS files can be carried out in any of a number of commercial software packages, and involves sequential “gating” or selection of populations of interest. For example, single cells might first be selected based on light scatter parameters; then live cells gated by exclusion of a viability dye; then lymphocytes identified by a combination of forward and side-angle light scatter; then T cells selected by expression of CD3; etc. Note that mass cytometry does not allow for light scatter properties to be measured, so all gating is done on the basis of labeled probes, in addition to cell length (as measured by the time duration of the cell’s ion cloud). Accurate quantitation of the proportions of even rare populations of cells can be made if enough events are collected; as can relative levels of expression of cellular proteins such as activation markers, intracellular cytokines, or signaling proteins. Absolute cell counts and quantitation of molecules of bound fluorophore/metal are also possible, for example, with reference to standardized labeled beads. Automated gating algorithms are also available, including unsupervised clustering and dimension reduction techniques [5–8].
Pre-conjugated antibodies are now readily available for both flow and mass cytometry, though the latter generally still requires a few conjugates to be made in-house, in order to complete a specific high-parameter panel. Sensitivity to detect low-abundance proteins can be an issue for both platforms. In general, the best fluorochromes have a detection limit of about 40 molecules per cell, while the detection limit for mass cytometry is about 400–500 molecules per cell. Sensitivity is influenced by factors such as autofluorescence (in traditional flow cytometry) and non-specifically bound antibodies (in both platforms). Compensation for spectral overlap can reduce sensitivity and introduce artifacts in fluorescence flow cytometry. In both platforms, there can be loss of cells in sample preparation washing steps, although mass cytometry generally requires more washing steps than fluorescence assays. Additionally, there are cell losses in the mass cytometer itself, such that data are captured on only about 30 % of introduced cells (improved to 50 % in the latest version of CyTOF instrumentation). Collection speed is also much lower in mass cytometry (300–500 events per second, compared to several thousand events/second in fluorescence flow cytometry).
Cells for these assays need to be in single-cell suspension. Debris and cell aggregates can interfere with the running of samples and with the interpretation of data. Because of the potential for cell loss, samples with <105 cells are usually not appropriate for either flow or mass cytometry. Functional assays, such as cytokine production, or analysis of phospho-signaling proteins, require cells with good viability. Overnight shipping of blood and cryopreservation of PBMC can compromise these readouts. Variability in sample handling, acquisition, and analysis are all significant in affecting results . Multiple approaches can mitigate these factors, from use of lyophilized reagents  to “barcoding” of samples to produce a single composite sample for purposes of uniform processing and acquisition [11–13].
Flow cytometry is backed by thousands of publications and over 30 years of development. Mass cytometry is more recent, but has seen an exponential rise in publications. Several flow cytometry assays are used in FDA-approved diagnostic tests. Both platforms can produce strong evidence, provided that appropriate controls are included.
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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