Biomedical Engineering Reference
In-Depth Information
points per day. Because each plate generates a single data file, the multiplex data must
be resolved. Moreover, there is a considerable variation in bead- and cell-based
assays. The cell-based assays vary according to the source and treatment of the cells.
With the creation of some 8 million data points over the course of the MLI pilot and
production phases, we have recognized the considerable effort required to balance the
needs of target development, HTS, and data analysis (“screening informatics”) for
plate-based flow cytometry. In particular, cell samples in HyperCyt do not always
resolve as well as the bead-based samples, so identifying individual wells has required
the development of automated binning algorithms in order that the increase in
throughput gained by sampling a plate as a file is not lost in data analysis.
Not unexpectedly, there is variability in sample preparation. In bead-based assays,
it is often possible to prepare plates in batches, so the plates for an entire day's screen
can be prepared consecutively, stored at the desired temperature, all independent of
data acquisition. In some cell-based targets, batches of 10 plates may be prepared with
screening interleaved. In some cases, cells can be fixed and batch analysis performed
on a separate day. In some cases, the cell plate preparation is timed and screening and
plate preparation must be coordinated. Finally, some cell-based incubations with
compounds are several days long, in which case batch preparation may precede HTS
on live cells by periods as long as a week during which the cells must retain their
ability to transit the flow cytometer. It is not surprising that strongly adherent cells
compromise convenient HTS.
Because UNMCMD functions in a production mode, with expected throughput of
6 targets per year for a library of 350,000 compounds, we are finding it useful to
incorporate the following operation elements:
4.5.1 Target Teams
Projects typically involve a target team, with a leader responsible for the screen and at
least one team member collaborating in the screen. UNMCMD target teams “own”
projects through the period of target development, HTS, dose-response follow-up,
and often through secondary assays and compound optimization.
4.5.2 Scheduling
We can interleave screens so that two screens are in process if necessary. Or we can
alternate days of HTS data acquisition and analysis. This may vary according to the
nature of the target. Three flow cytometers are available for the primary screen along
with dedicated sample preparation automation. A fourth flow cytometer is available
along with a secondary sample preparation station for target development and
implementation prior to HTS and for dose-response assays, when the primary
instrumentation should be in use for another project.
4.5.3 Accessibility
Integration of HyperCyt has been achieved with essentially all commercial flow
cytometry systems. However, consideration needs to be given to the geometry of the
Search WWH ::
Custom Search