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Laboratory and Clinical Utility of Mass Cytometry

December 21, 2021

January 2022

A recent innovation, mass cytometry is a variation of flow cytometry that is gaining momentum. Flow cytometry is a now ubiquitous method, and the gold standard, for identifying types and classes of cells in laboratory research, as well as in patients’ samples, including for the diagnosis, prognosis, and monitoring of hematologic malignancies and other disorders.

In flow cytometry, cell surface markers are identified with antibodies that are linked to a fluorophore, a fluorescent chemical compound that is excited by lasers within the flow cytometer, after which the fluorophore reemits light. The reemitted light energy is then converted into electronic signals that are analyzed by a computer.

Flow cytometers can be used to analyze the cellular makeup of a mixed population of cells and for cell sorting with the goal of producing a pure population of cells for cell lines, stem cells, or cloning. Flow cytometry enables single-cell level analyses, which can be used to determine what an experimental drug does to individual cells. The technique is quite fast, producing data on about 10,000 cells per second. Standard flow cytometry machines can detect a combination of about eight to 10 cell surface markers on a cell in a single experiment, and high-end cytometers can now identify up to 20 markers.

“Mass cytometry is like doing flow cytometry, but with the ability to detect as many as 50 markers on and inside a cell,” said Gregory K. Behbehani, MD, PhD, a hematologist-oncologist and assistant professor of internal medicine at The Ohio State University, where he runs a research laboratory in which mass cytometry is used for single-cell deep functional phenotyping. “You get back similar types of data; the concepts of the data analysis and experimental design are very similar, but the technical aspects of the two techniques are very different.”

ASH Clinical News spoke with Dr. Behbehani and Stephen Oh, MD, PhD, associate professor of hematology and co-head of the Immunomonitoring Laboratory at the Washington University School of Medicine in St. Louis, about the principles of mass cytometry, how it differs from flow cytometry, and how researchers and clinicians are applying this technique in the laboratory and clinic.

Mass Cytometry 101

Mass cytometry is a cytometric technique that is like fluorescence flow cytometry in that both methods detect antibodies binding to cell-expressed antigens. A key difference between flow and mass cytometry is that in mass cytometry, the fluorophore is replaced by an isotopically purified heavy metal atom, which is not normally found in biological systems. The antibodies used for both techniques are the same, conjugated either to a fluorophore or a heavy metal molecule. Detection of the expression of any protein inside or on the surface of a cell is essentially limited only by the availability of an antibody that can specifically bind the cellular protein of interest.

Because many heavy metals and their isotopes are available, mass cytometry has the advantage of being able to analyze as many as 50 cellular markers. The presence of the bound antibodies is detected by inductively coupled plasma ionization and a time-of-flight mass spectrometry analysis of the metal ions that were attached to each antibody.

“The benefit of using the heavy metals is that many of these can be detected simultaneously by the mass spectrometer whereas with flow cytometry, autofluorescence and overlapping fluorescent signals limit the number of parameters that can be measured simultaneously in a single cell,” Dr. Oh explained.

In contrast, the mass spectrometer can distinguish very small differences in the atomic weights of different ions without overlap. “The large number of markers that can be analyzed in a single experiment is a huge advantage, especially for complex, heterogenous cell populations that can be difficult to analyze with flow cytometry,” Dr. Behbehani noted.

To conduct the analysis, the cells are initially permeabilized and mixed with the appropriate number of antibodies, each conjugated to a distinct rare earth metal or isotope. In the cytometer, the cells bound to the antibodies are vaporized and the electrons are stripped off the metals to generate ions. The metal ions are then put through an electromagnetic field and a detector can register the different isotopes, counting how much of each distinct metal is present and, in turn, the number of its antigen targets.

Among the initial descriptions of mass cytometry was by Olga Ornatsky, Scott D. Tanner, and their colleagues from the University of Toronto in 2008.1 This initial study was followed by many others, including a 2011 study by Sean Bendall, PhD, and Garry Nolan, PhD, of Stanford University, and their colleagues.2 This 2011 study demonstrated the ability to simultaneously measure 34 distinct parameters in single cells from healthy human bone marrow, including binding of 31 antibodies, cell viability, DNA content, and relative cell size.

Like flow cytometry, mass cytometry is quantitative and provides researchers with the ability to perform multiplexed single-cell measurements. The disadvantages of mass cytometry include that the sample processing is slower – about 300 to 500 cells can be accurately analyzed per second – and the sample is destroyed during analysis, ruling out the possibility of cell sorting. Another is that antigens that are in very low abundance may be difficult to detect, although researchers are working on techniques to amplify these weak signals.

A major advantage of mass cytometry is the number of cellular markers that can be detected. While mass cytometers currently detect up to 50 markers in a single experiment, the machine could theoretically measure up to 120 markers, as long as there are enough isotopically pure heavy metals accessible that could be attached to antibodies.

Beyond Cell Surface Markers

“I help run our core facility, and one of the big debates is when deciding whether to use flow or mass cytometry,” Dr. Behbehani said. “The way I see it, flow cytometry is for the basic – up to 10 cell-surface-marker experiments. Where mass cytometry will be used is for the situations when you want to go beyond the cell surface and look at intracellular signaling, cytokines, transcription factors, and other markers on the inside of cells and at cell cycle state.”

With flow cytometry, permeabilizing the cells can be incompatible with fluorophore fluorescence detection, making intracellular marker detection tricky or impossible.

“A big advantage of mass cytometry is that the antibody-heavy metal molecules are really tough and can go through anything and look essentially the same,” Dr. Behbehani explained. “There are a lot of things that can subtly change fluorescence, but almost nothing that can change the atomic mass of a metal, so mass cytometry is much more robust for studying proteins inside the cell and surface and intracellular markers simultaneously.”

To study intracellular signaling, there may not be a suitable and available antibody for every protein of interest, Dr. Oh noted, but that is also the case with flow cytometry.

To study the cell cycle, 5-iodo-2′-deoxyuridine (IDU) can be incorporated into the DNA instead of thymine. The iodine is then attached and detected directly by its mass to identify cells that are replicating.3

In 2015, Dr. Behbehani and colleagues applied this technique to analyze the proportion of cells from bone marrow aspirates of patients with acute myeloid leukemia (AML) that is quiescent and actively replicating.4 They were testing the hypothesis that patients with AML who have a high relapse rate tend to have a larger proportion of quiescent leukemia stem cells (LSCs) that are refractory to chemotherapy. Using 41 patient samples and five healthy donor samples, the team found that patients with clinically favorable AML had a five-fold higher fraction of cells in S-phase compared to samples from patients with clinically unfavorable AML. The mass cytometry analyses also allowed direct observation of the in vivo effects of cytotoxic chemotherapy on the leukemic cells. This was the first direct evidence supporting the hypothesis that patients with refractory AML have a higher proportion of non-replicating LSCs that are refractory to chemotherapy.

“Basically, our data . . .  suggests that one of the reasons so-called ‘good-risk AML’ has a good prognosis is that the stem cells are more proliferative and, therefore, can be killed with cytotoxic chemotherapy. In contrast, chemotherapy doesn’t work on higher-risk disease because these cells are not dividing and [are] refractory to the therapy,” Dr. Behbehani said.

Dr. Oh and colleagues have used mass cytometry to profile the signaling pathways activated in myelofibrosis. They showed that myelofibrosis results in constitutively hyperactive nuclear factor (NF) kappa B signaling that contributes to the disease.5  In a subsequent study, Dr. Oh and colleagues analyzed the cytokines present in myelofibrosis patient samples and showed that many cytokines are constitutively overproduced as a result of the NF kappa B and also mitogen-activated protein kinase signaling in myelofibrosis.6 Most recently, the team found that the drug pevonedistat could target the NF kappa B pathway and inhibit growth of malignant cells in myeloproliferative neoplasm patient samples, providing support to test pevonedistat in patients with myelofibrosis.7

Clinical Utility

“I have long advocated for mass cytometry to be used in the clinic,” Dr. Behbehani said. Currently, however, mass cytometry is used primarily for research, including on patients’ samples and in clinical trials, but not necessarily to provide real-time information for patient care or to guide therapy decisions for those patients not on a clinical trial.

“We’re working on a paper now where we compared clinical flow cytometry results with mass cytometry results,” Dr. Behbehani said. “We show that we can get the same blast count with both techniques with a correlation factor of over 90%. Basically, we can recreate the data that the clinical flow cytometry lab generated and used to make a patient diagnosis. Had we used mass cytometry, we would have correctly diagnosed these patients.”

Both Drs. Oh and Behbehani see mass cytometry being used to better understand the immune system and generate data on how the immune system of a patient with cancer changes in response to an immunotherapy, whether the treatment is working, and what subsequent therapy is appropriate.

There are also clinical trials that are using mass cytometry to analyze patient samples to understand whether the investigational drug is hitting its target – data that could, in the future, also be translated to the clinic to understand whether an approved drug is working for a patient.

The current barriers to mass cytometry analyses in the clinic are designing and setting up the panel of markers. “Many experiments are started from scratch, so it takes months to set up and execute,” Dr. Behbehani said. Once the initial setup is complete, however, patients’ samples could be analyzed and interpreted within three to five days, he added.

In the not too distant past, few cancer centers even had a mass cytometer, but this may be changing.

“Five years ago, we would say all the cool kids had one,” Dr. Behbehani said. “Now, many of the other kids have one, too.”

—By Anna Azvolinsky


  1. Ornatsky OI, Lou X, Nitz M, et al. Study of cell antigens and intracellular DNA by identification of element-containing labels and metallointercalators using inductively coupled plasma mass spectrometry. Anal Chem. 2008;80(7):2539-2547.
  2. Bendall SC, Simonds EF, Qiu P, et al. Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science. 2011;332(6030):687-696.
  3. Devine RD, Behbehani GK. Use of the pyrimidine analog, 5-iodo-2′-
    deoxyuridine (IdU) with cell cycle markers to establish cell cycle phases
    in a mass cytometry platform. J Vis Exp. 2021;176:e60556.
  4. Behbehani GK, Samusik N, Bjornson ZB, Fantl WJ, Medeiros BC, Nolan GP. Mass cytometric functional profiling of acute myeloid leukemia defines cell-cycle and immunophenotypic properties that correlate with known responses to therapy. Cancer Discov. 2015;5(9):988-1003.
  5. Fisher DAC, Malkova O, Engle EK, et al. Mass cytometry analysis reveals hyperactive NF Kappa B signaling in myelofibrosis and secondary acute myeloid leukemia. Leukemia. 2017;31(9):1962-1974.
  6. Fisher DAC, Miner CA, Engle EK, et al. Cytokine production in myelofibrosis exhibits differential responsiveness to JAK-STAT, MAP kinase, and NFκB signaling. Leukemia. 2019;33(8):1978-1995.
  7. Kong T, Laranjeira ABA, Collins T, et al. Pevonedistat targets malignant cells
    in myeloproliferative neoplasms in vitro and in vivo via NFkB pathway inhibition [published online ahead of print, 2021 Oct 13]. Blood Adv. doi:10.1182/bloodadvances.2020002804.


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