Immune monitoring technology primer: flow and mass …
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SHORT REPORT Open Access Immune monitoring technology primer: flow and mass cytometry Holden T. Maecker 1* and Alexandre Harari 2 Description of the technology Flow cytometry traditionally uses fluorochrome-labeled probes, such as antibodies, to identify cells expressing the targets of those probes. A sample stream carries single cells in suspension past a laser, exciting the fluorochromes, with quantitation of the emitted fluorescent signals from each cell via optical filters and photomultiplier tubes [1]. In mass cytometry, or CyTOF, the fluorescent labels are replaced with heavy metal ions. The metal ions are che- lated to a polymer, which is covalently linked to antibodies or other probes. After staining with these probes, single cells are introduced via an aerosol stream into a plasma torch, resulting in complete ionization of the labeled cells. The heavy ions are then focused via a quadrapole and enter a time-of-flight detector, where the individual ions are quantitated [2, 3]. This results in the simultaneous benefit of more available labels, with much less spillover between detector channels (Fig. 1). Type of data obtained/readout Flow and mass cytometry uniquely allow for the quantita- tion 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 [4]. It is not unusual to collect data on 10 5 –10 7 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 com- bination 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. Ab- solute cell counts and quantitation of molecules of bound fluorophore/metal are also possible, for example, with ref- erence to standardized labeled beads. Automated gating algorithms are also available, including unsupervised clus- tering and dimension reduction techniques [5–8]. Limitations of the approach 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. Sensi- tivity 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 mol- ecules per cell. Sensitivity is influenced by factors such as autofluorescence (in traditional flow cytometry) and non- specifically bound antibodies (in both platforms). Com- pensation 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). * Correspondence: [email protected] 1 Institute for Immunity, Transplantation, and Infection, Stanford University School of Medicine, Stanford, CA, USA Full list of author information is available at the end of the article © 2015 Maecker and Harari. 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. Maecker and Harari Journal for ImmunoTherapy of Cancer (2015) 3:44 DOI 10.1186/s40425-015-0085-x on November 21, 2021 by guest. Protected by copyright. http://jitc.bmj.com/ J Immunother Cancer: first published as 10.1186/s40425-015-0085-x on 15 September 2015. Downloaded from
Transcript of Immune monitoring technology primer: flow and mass …
SHORT REPORT Open Access
Immune monitoring technology primer:flow and mass cytometryHolden T. Maecker1* and Alexandre Harari2
Description of the technologyFlow cytometry traditionally uses fluorochrome-labeledprobes, such as antibodies, to identify cells expressing thetargets of those probes. A sample stream carries singlecells in suspension past a laser, exciting the fluorochromes,with quantitation of the emitted fluorescent signals fromeach cell via optical filters and photomultiplier tubes [1].In mass cytometry, or CyTOF, the fluorescent labels arereplaced with heavy metal ions. The metal ions are che-lated to a polymer, which is covalently linked to antibodiesor other probes. After staining with these probes, singlecells are introduced via an aerosol stream into a plasmatorch, resulting in complete ionization of the labeled cells.The heavy ions are then focused via a quadrapole andenter a time-of-flight detector, where the individual ionsare quantitated [2, 3]. This results in the simultaneousbenefit of more available labels, with much less spilloverbetween detector channels (Fig. 1).
Type of data obtained/readoutFlow and mass cytometry uniquely allow for the quantita-tion of multiple parameters on many individual cells. Upto 17 or more parameters are possible with fluorescence,while 40 or more parameters can be quantitated with masscytometry [4]. It is not unusual to collect data on 105–107
cells per sample by either technique. The data from eachsample is compiled in a Flow Cytometry Standard (FCS)file, for both flow and mass cytometry. The FCS file liststhe intensities obtained for each probe on each individualcell. Analysis of FCS files can be carried out in any of anumber of commercial software packages, and involvessequential “gating” or selection of populations of interest.For example, single cells might first be selected based onlight scatter parameters; then live cells gated by exclusionof a viability dye; then lymphocytes identified by a com-bination of forward and side-angle light scatter; then T
cells selected by expression of CD3; etc. Note that masscytometry does not allow for light scatter properties to bemeasured, so all gating is done on the basis of labeledprobes, in addition to cell length (as measured by the timeduration of the cell’s ion cloud). Accurate quantitation ofthe proportions of even rare populations of cells can bemade if enough events are collected; as can relative levelsof expression of cellular proteins such as activationmarkers, intracellular cytokines, or signaling proteins. Ab-solute cell counts and quantitation of molecules of boundfluorophore/metal are also possible, for example, with ref-erence to standardized labeled beads. Automated gatingalgorithms are also available, including unsupervised clus-tering and dimension reduction techniques [5–8].
Limitations of the approachPre-conjugated antibodies are now readily available forboth flow and mass cytometry, though the latter generallystill requires a few conjugates to be made in-house, inorder to complete a specific high-parameter panel. Sensi-tivity to detect low-abundance proteins can be an issue forboth platforms. In general, the best fluorochromes have adetection limit of about 40 molecules per cell, while thedetection limit for mass cytometry is about 400–500 mol-ecules per cell. Sensitivity is influenced by factors such asautofluorescence (in traditional flow cytometry) and non-specifically bound antibodies (in both platforms). Com-pensation for spectral overlap can reduce sensitivity andintroduce artifacts in fluorescence flow cytometry. In bothplatforms, there can be loss of cells in sample preparationwashing steps, although mass cytometry generally requiresmore washing steps than fluorescence assays. Additionally,there are cell losses in the mass cytometer itself, such thatdata are captured on only about 30 % of introduced cells(improved to 50 % in the latest version of CyTOFinstrumentation). Collection speed is also much lower inmass cytometry (300–500 events per second, compared toseveral thousand events/second in fluorescence flowcytometry).
* Correspondence: [email protected] for Immunity, Transplantation, and Infection, Stanford UniversitySchool of Medicine, Stanford, CA, USAFull list of author information is available at the end of the article
© 2015 Maecker and Harari. Open Access This article is distributed under the terms of the Creative CommonsAttribution 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 thesource, provide a link to the Creative Commons license, and indicate if changes were made. The Creative CommonsPublic Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data madeavailable in this article, unless otherwise stated.
Maecker and Harari Journal for ImmunoTherapy of Cancer (2015) 3:44 DOI 10.1186/s40425-015-0085-x
on Novem
ber 21, 2021 by guest. Protected by copyright.
http://jitc.bmj.com
/J Im
munother C
ancer: first published as 10.1186/s40425-015-0085-x on 15 Septem
ber 2015. Dow
nloaded from
Types of samples needed and special issuespertaining to samplesCells for these assays need to be in single-cell suspen-sion. Debris and cell aggregates can interfere with therunning 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 masscytometry. Functional assays, such as cytokine produc-tion, or analysis of phospho-signaling proteins, requirecells with good viability. Overnight shipping of bloodand cryopreservation of PBMC can compromise thesereadouts. Variability in sample handling, acquisition, andanalysis are all significant in affecting results [9]. Mul-tiple approaches can mitigate these factors, from use oflyophilized reagents [10] to “barcoding” of samples toproduce a single composite sample for purposes of uni-form processing and acquisition [11–13].
Level of evidenceFlow cytometry is backed by thousands of publicationsand over 30 years of development. Mass cytometry ismore recent, but has seen an exponential rise in publica-tions. Several flow cytometry assays are used in FDA-approved diagnostic tests. Both platforms can producestrong evidence, provided that appropriate controls areincluded.
Competing interestsThe authors declare that they have no competing interests.
Authors’ contributionsHTM drafted the manuscript and AH added to and edited it. Both authorsread and approved the final manuscript.
Author details1Institute for Immunity, Transplantation, and Infection, Stanford UniversitySchool of Medicine, Stanford, CA, USA. 2Centre Hospitalier UniversitaireVaudois (CHUV), Epalinges, Switzerland.
Received: 31 July 2015 Accepted: 6 August 2015
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Fig. 1 a Example of emission spectra of several dyes used in fluorescence flow cytometry, showing the degree of overlap and consequentspillover between detectors. b Ion signals detected by mass cytometry are by comparison very discrete, allowing many more simultaneousprobes to be used, with little or no spillover
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