In some cases you may want to flag certain wells and/or analytes as unreliable, in order to exclude the unreliable data from later analysis.
A replicate group is a set of wells that all contain the same sample at the same dilution. For example, the replicate groups in the sample data file used in this tutorial each encompass two wells. Each pair of wells contains the same unknown sample at the same dilution. In the data file, wells that are part of a replicate group are listed in sequential rows and have the same value in the "Type" column; for example, the two wells with a "Type" of "X1" are part of one replicate group.
For example, you might wish to exclude an analyte's results for all sample wells in a replicate group because fluorescence intensities reported for that analyte for that group had high coefficients of variation. Alternatively, you might wish to exclude all measurements for the entire run for a particular analyte after discovering that this analyte was bound to beads from a defective lot.
Here, you will exclude a single analyte for all sample wells within a replicate group, in this case there are two wells per group.
In this example scenario, since the fluorescence intensity reported for an analyte in a well is the median for all beads bound to that analyte in that well, you might exclude the analyte if the reported coefficient of variation for the group was unusually high.
Filter the results grid so that you see only unknowns, not background, standard or control wells.
Notice that the grid now shows your new filter, as well as a filter to show only the specific run. Before continuing to the next step of modifying the default grid view, clear the run filter by clicking the 'X' (your run number will likely be different). Otherwise the default grid view will only show this particular run going forward.
Customize the results grid to show instrument-reported coefficients of variation for each well:
To exclude data for a replicate group, click the (circle/slash) icon for a row in the group. Select the replicate group using the checkbox, then choose whether all analytes for the sample group are excluded, or just the row you picked initially.
To undo a replicate group exclusion, you would again click on the (circle/slash) icon for the excluded row, then uncheck the relevant analytes in the popup and click Save again.
Once excluded, the rows are color coded with red/pink and the Flagged As Excluded field changes value from "no" to "yes". Use this field to sort and filter the assay data, if desired.
You can also see an example in the interactive example.
For additional detail about why a given row is marked as excluded, you can customize the grid view to include the Exclusion Comment field. This can help you identify whether the row is excluded as part of a well group, analyte exclusion, etc.
Next, we exclude a given analyte for all wells in the run. For example, you might wish to exclude all measurements for a particular analyte if you realized that this analyte was bound to beads that came from a problematic lot.
To remove an analyte exclusion, select Exclusions > Exclude Analytes again. In the popup dialog, clear any exclusion checkmarks, and click Save.
When you exclude data from a well-group within a titration, the assay is re-calculated (i.e. the transform script is rerun and data is replaced). If you want to exclude all well groups for a given titration, you can do so without recalculating by selecting Exclusions > Exclude Titration and specifying which titration to exclude. Note that you cannot exclude a "Standard" titration.
The exclusion popup in this case lets you select each titration present and check which analytes to exclude for that titration. Note that analytes excluded already for a replicate group, singlepoint unknown, or at the assay level will not be re-included by changes in titration exclusion.
To exclude analytes from singlepoint unknown samples, select Exclusions > Exclude Singlepoint Unknowns. The unknown samples are listed, and you can select each one in turn and choose one or more analytes to exclude from only that singlepoint.
Note that analytes excluded for a replicate group, titration, or at the assay level will not be re-included by changes in singlepoint unknown exclusions.
When you open the UI to exclude a replicate group, the default option is to only exclude the single well for the row you selected. You can choose to exclude all analytes or only selected analytes for that well.
Click View Excluded Data above the results grid to see all exclusions in a single page. This might help you see which data needs to be re-run. You could of course view excluded rows by filtering the grid view on the Flagged as Excluded column, but the summary page gives a summary of exclusions across multiple runs.
If you are looking at the results grid for a single run, the View Excluded Data report will be filtered to show the exclusions for that run.
You can see a similar view in the interactive example.
Return to the list of runs by clicking View Runs. If you wanted to reimport the run, perhaps to recalculate positivity, you could do so by selecting the run and clicking Reimport Run. Note that the previous run data will be deleted and overwritten with the new run data.
You will see the same entry screens for batch and run properties as for a new run, but the values you entered previously will now appear as defaults. You can change them for the rerun as necessary. If exclusions have been applied to the run being replaced, the analyte properties page will include an Exclusion Warning panel.
The panel lists how many exclusions are applied (in this case one replicate group, one analyte, and one single point control). Click Exclusions Report to review the same report as you saw using the "View Excluded Data" link from the run data grid. Check the Retain matched exclusions box to retain them; then click Save and Finish to initiate the reimport.
If any exclusions would no longer match based on the reimport run data, you will see a red warning message saying the exclusion will be lost if you proceed with the import.
[ Video Overview: Retain Luminex Exclusions on Reimport ]
When the reimport is complete, click the name of the run to see the reimported data, with any exclusions you chose to retain.
To edit an exclusion, reopen the panel where you originally added the exclusion at that level. Adjust the settings and save. As with the original exclusion, this operation will run in the background.
To remove an exclusion, edit it and remove all selections before saving.
This happens only when your assay design is associated with a transform script (see: Luminex Assay Tutorial Level II). When you exclude an entire replicate group from a titrated standard or quality control, LabKey Server automatically re-runs the transform script for that run.
This is desirable because changing the replicates included in a titration affects the calculations that the script performs for the curve fits and other measure (e.g., EC50, AUC, HighMFI, etc., as defined in Luminex Calculations).
If you exclude a replicate group that is not part of a titration (e.g., part of an unknown), the calculations performed by the script will be unaffected, so the script is not re-run.
During data import, LabKey Server adds a quality control flag to each data row where reported FI is greater than 100 and %CV (coefficient of variation) is outside of a certain threshold. For unknowns, the %CV must be greater than 15 to receive a flag; for standards and quality controls, the %CV must be greater than 20.
Columns flagged through this process are highlighted in red. If flagging is later disabled, the row is no longer highlighted.
To see all rows that received flags according to these threshholds, you can add the CVQCFlagsEnabled column to a Luminex data or results view and filter the data using this column. The column is hidden by default.
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