FLOW CYTOMETRY IN PRECLINICAL TOXICOLOGY/SAFETY ASSESSMENT - Flow Cytometry in Drug Discovery and Development - page 142

evidence of this at present. In addition, there is a small percentage of coincident or
aggregate events that also must be excluded in analysis of flow cytometry samples,
but this seems unlikely to negatively impact the overall differential as cells excluded
as aggregates are preferentially not one cell type. It should be noted that the
exclusion of cell aggregates is a common practice in flow cytometric analysis and is
performed to assure that data are from individual cells.
The possibility that preparation of mouse bone marrow samples for flow cyto-
metric analysis leads to preferential removal and/or destruction of erythroid cells is
another possibility that cannot be discounted. In order to have full confidence in
flow cytometric results from mouse bone marrow, the lack of parity between flow
cytometry and manual cytology would need to be resolved.
6.8 SUMMARY
The termhigh-content screening (HCS) or cell-based screening plays amajor role in
drug discovery and development in a pharmaceutical company and was originally
employed in bioimaging-based systems. As flow cytometry technology has evolved
to provide expanded color capabilities, the technology has become both a robust and
an economic cell-based HCS system assaying thousands of cells per second. Flow
cytometry provides several key advantages over bioimaging systems. First, this
higher single-cell throughput technology allows researchers to analyze smaller
population subsets with greater statistical accuracy. Second, the cell sorting can be
used to collect theseminor cell populations for secondarymolecular testing, gaining
additional data for understanding the mechanism and kinetics.
While flow cytometry provides advantages during the preclinical toxicity
screening process (e.g., more information in less time), there are also challenges.
First, the researchers must develop the assay. In the case of the oxidative stress model,
a thorough titration of the labels was needed to optimize the assay. Any change in cell
line or culture conditions would likely require similar titrations to optimize the assay.
Biomarker assay development requires the availability of the appropriate antibodies
and substrates to label key biomarkers. As was discussedwith the CEC/CEP assay, the
process may be one of elimination as well (gating out of the platelets, Tand B cells that
shared the CD36 marker). The development process requires a detailed knowledge
of cell lineage markers, intracellular processes, and access to labeled antibodies/
substrates for evaluation of thesemarkers and processes. Functional substrates such as
those employed in the oxidative stress study may have a fixed color or may be
available inmodified forms to allow selection of a “convenient” color during the assay
development.
Second, the system must be tested with compounds that have a well-
characterized toxicity profile (e.g., serotonin treatment with rats) and the out-
comes must be validated with information from well-known secondary testing
outcomes (e.g., gene or protein expression studies). The sorted CD36 þ /Lin
cells were tested for both culture characteristics and gene expression and showed