Biomedical Engineering Reference
In-Depth Information
15.7.3 Advantages of the Mass Cytometry Approach
15.7.3.1 High Dimensionality Using the current set of reagents, the ability to
measure 30 parameters simultaneously, as compared to the maximum of 17 reported in
fluorescence, offers a number of new avenues in cytometry analysis. More detailed
characterization based on cell surface antigen expression of immune and other
hematopoetic cell types are some of the first obvious experiments. In addition,
simultaneous measurement of intracellular antigens (e.g., proteins) and their
modifications (e.g., phosphorylation) that regulate cell cycle, apoptosis, and gene
expression in rare cell populations (e.g., hematopoetic stem cells) becomes feasible.
This type of measurement is particularly difficult to do in greater than 10 dimensions by
fluorescence because many of the changes detected are subtle and can suffer heavy
interference from spectral overlap. The lack of compensation combined with high
dimensionality in mass cytometry should enable the measurement of more than 10
intracellular parameters while still allowing simultaneous measurement of surface
antigen expression. In preliminary experiments, we have already been able to use the
CyTOF mass cytometer to perform phosphorylation analysis on 17 parameters
simultaneously (Figure 15.11). This high-dimensional phosphorylation analysis will
also aid in the creation of better profiles for clinical diagnostics and will further our
ability to evaluate target and off-target effects simultaneously during drug screening.
15.7.3.2 Simplicity/Versatility Because the mass spectrometer provides at least
three orders of magnitude resolution between adjacent detection channels, meaning
that two adjacent metal isotopes can differ in abundance by 10 3 before spectral
overlap becomes an issue, compensation is only minimally required, and in most
instances it is not required at all. In addition, the signal responses for the current
commercial metal isotope tags are approximately twofold of one another on the
CyTOF mass cytometer. Taken together, these attributes indicate that tag-panel
selection can essentially be arbitrary, making experimental design much simpler.
Consequently, even if a user does not have 30 parameters to interrogate, the design of
staining panels for as few as 10 parameters could be accomplishedmore rapidly than it
can for fluorescence-based experiments.
15.7.3.3 Stability The elemental tags have an added advantage over many
fluorescent reagents in that they are extremely stable chemically. So unlike several
fluorophores, such as the phycobiliproteins (PE and APC), they remain unaffected
during procedures such as cell permeabilization with methanol or other strong
denaturants. Elemental tags will be stable under these conditions. Characteristics
such as this are essential in intracellular phosphoprotein analysis.
15.7.3.4 Speed/Throughput Although the current throughput is limited to about
1000 cells per second, the large number of parameters that can be measured
simultaneously by mass cytometry provides a dramatic increase in the data
acquisition rate. For example, a simultaneous 30 parameter single-cell analysis
would require multiple samples with overlapping parameters on a fluorescence
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