Biomedical Engineering Reference
In-Depth Information
develop a biomarker that requires minimal invasive tissue collection in order to
provide the opportunity for development as a clinical biomarker as well. Flow
cytometric analysis of blood often holds the potential to provide such a biomarker.
6.3 CELL SORTING AND HIGH CONTENT ANALYSIS
Investigative toxicology may also fall under the purview of preclinical toxicology
departments as a separate unit or as a portion of responsibility. Investigative studies
utilizing flow cytometry can be done ex vivo or in vitro to answer relatively simple
questions of relative toxicity or mechanism. For example, a multiparameter analysis
of oxidative stress could be used as a screening tool for multiple compounds in a drug
chemical series to aid in candidate selection for in vivo experiments. Another example
is using a calcium flux analysis on cardiomyocytes to determine if the mechanism of
cardiotoxicity is due to perturbation of calcium channels. There are many, many ways
to apply flow cytometry to investigative toxicology.
All applications described thus far deal only with simple flow cytometric analysis.
But there is further potential for application of cell sorting. If, as described, a single-
cell suspension can be achieved for analysis by flow cytometry, then cells of interest
can be isolated by cell sorting for further experimentation or analysis. Examples of
postsort applications include functional assays, cell culture, gene expression analysis,
and imaging of enriched cell populations of interest. Cell sorting with subsequent
gene expression analysis can be very powerful and provide data unobtainable in any
other manner. An example is gene expression analysis of a rare cell population whose
transcriptional profile is buried in the “noise” created by more prevalent cell types.
Similarly, enrichment of rare target cells for subsequent culture or functional assays
provides an opportunity that is nonexistent when these cells are in a heterogeneous
mixture. Even if the cells are not rare, isolation of cells of interest eliminates an
inherent flaw in gene expression (i.e., mRNA) analysis of mixed populations. This is
due to the fact that an increase in the amount of transcript measured, normalized to
some housekeeping gene, is universally interpreted as an increase in transcription,
often stated as “upregulation,” of a gene of interest. But in an uncharacterized
heterogeneous population, this could just as likely be due to an increase in percentage
of cells expressing the said gene. Assuming all cells in the starting heterogeneous
mixture contain roughly equal amounts of mRNA, enrichment of the cell population
of interest to just 90% purity is sufficient for providing a valid transcription
signature [1]. A case where this assumption is invalid is isolation of reticulocytes
or platelets from whole blood where just a few leukocytes per million target cells can
greatly skew results since leukocytes contain a two to three orders of magnitude
greater amount of mRNA.
Application of flow cytometric analysis in preclinical toxicity studies provides
multiple benefits. Multiparameter immunophenotyping of blood or bone marrow, for
example, provides a very rich data set compared to simple hematologic analysis.
Instead of simply gaining information about the major hematopoietic populations,
data on subpopulations, activation states, proliferation, cell cycle, state of maturation,
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