...is the assay calculation and data review application of the ScreenAble toolkit. Because of its central role in the screening process, the transactional objects of this application, AssayRuns, are the units of production for the screening process. AssayRuns are defined as a group of plates prepared from the same batch of reagents and assayed as a group.

AssayRuns are derived from PlateRuns and inherit their Properties and Methods. Additional parameters are added to the PlateRun object in the form of an AssayProtocol and, for production runs, SamplePlates of test substances. Data structures suitable for holding Screen parameters, normalized Results and derived Calculated Results are also added to the run object according to the class of AssayRun being created(see below). Once calculated, the run may be navigated and examined using the abundant plot and summary tools of the application.

Data Review in ScreenAble

Options for data review include setting the Pass / Fail status for selected plates. An AssayRun's results cannot be uploaded unless all plates have a review status. The interface includes several key control chart options to assist in this review and provide for rapid idenfication of problem data issues.

If a plate is failed, its well data is sent to a results table separate from the passed plates. Individual control wells may be addressed using a different mode of the same tool. This may involve the removal of controls with errors and a re-calculation of plate activity as appropriate. Comments may be attached to selected plates or control wells.

Potency Curve Review

Select calculated result fits can be appreciated and reviewed in the interface shown here. The nls() function of R is used to fit parametric models of dose responsiveness. In addition to the recommended 4-parameter-logistic fit, 3-parameter fixed top or bottom fits and interpolated curves are also computed and can be chosen as alternate fits.

Users may "drop" outlier well values using the mouse or data checkboxes and "refit" the data as needed. The contents of interface are determined by the selected rows of the CalcResults datagridview (all are used if none selected). Sort order of the CalcResult data is respected and can be used to organize fits for review e.g., sort by RSQ ascending brings poor fits to top.

Calculation Classes in ScreenAble

• Single - a single value is calculated for each sample well and normalized according to the assay window defined by Top and Bottom controls. This mode is used for broad general screening of large sample collections.
• Replicate - in addition to single calculations, a single mean or median value is calculated for the replicated sample wells. Simple replication allows for increased accuracy of determinations in situations where rank order potency of a set of samples at a single concentration is desired.
• Potency (PotencyRep) - in this mode samples are serially diluted across either axis of the assay plate or through the set of plates in order to establish the potency of compounds determined to be active in the Single mode. Parametric fits or interpolations are used to determine an IC50 or other desired measure of potency. The parameters and their error terms are stored in the database.
• Mechanistic - this mode uses the multi-dimensional plate layouts of ScreenAble to perform sophisticated steady-state kinetic calculations to determine both potency and mechanism of action for test substances.