Biomedical Engineering Reference
In-Depth Information
potential drug targets. In this chapter, we describe the application of phospho
flow to analysis of samples from murine models of systemic lupus erythema-
tosus (SLE) and from human patients.
High-Throughput Screening Flow cytometry is often used for in-depth analysis
of a small number of samples, perhaps for secondary screening. Here we will
discuss fluorescent cell barcoding (FCB), a technique that multiplexes samples
to dramatically improve sample throughput and reduce reagent consumption.
This technique, coupled with modern hardware for sample acquisition, makes it
feasible to run thousands of samples per day, providing a platform for screening
of focused libraries of tens of thousands of compounds.
Secondary Screening/Preclinical Testing Once lead compounds are identified,
they must be tested in animal models. Here, the same assays that were used to
first understand the disease and identify the target can again be used to
determine if the compound is selective for the pathway and cell type of interest.
In this way, no new assays must be developed to study the mechanism of drug
action. In this chapter, we will discuss the search for inhibitors of cytokine-
induced Janus kinase-signal transducer and activator of transcription (Jak-Stat)
signaling pathways and the testing of these molecules in vivo. Importantly,
analysis of multiple cell types allowed us to identify potent drugs that would
otherwise have been overlooked because they affect only a small proportion of
the total peripheral blood cells. In addition, we will show how a “druggability
landscape” can be developed based on observations of how multiple com-
pounds inhibit particular cellular subsets and signaling pathways.
Patient Stratification and Diagnostics Drug therapies can only be effective if a
patient has the appropriate drug target or modifications to their intracellular
signaling networks. Examples from cancer research have shown that certain
therapies should only be used in patients who overexpress particular antigens.
In this chapter, we will discuss patient stratification in acute myeloid leukemia
(AML). By analysis of signaling networks of individual patients, it was possible
to stratify patients into groups that were either responsive or nonresponsive to
standard treatments. This type of analysis allows important diagnostic decisions
to be made and treatments to be tailored to individual patients. The ability to
monitor changes in multiple cellular subsets enables a detailed view of disease
and allows early identification of patients who will not respond or who will
relapse after treatment.
Next-Generation Flow Cytometry The multiparameter capabilities of the flow
cytometer enable a unique view of drug action and disease analysis. Current
cytometers, based on fluorescence, can measure up to 17 parameters per cell.
However, in practice, making this many measurements is limited by the
presence of fluorescence emission compensation, where signals from one
fluorophore emit into multiple detector channels, convoluting analysis. Here
we discuss a new platform, called mass cytometry, which is based not on
fluorescently tagged antibodies but on metal isotope-tagged antibodies. Cells
are introduced into a mass spectrometer and the quantity of each of the metal
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