Pre-processing and quality control of the data is performed using the Olink® NPX Manager software – see the NPX Manager product page for more information.
The built-in quality control (QC) allows us to have control over the technical performance of the assays as well as the samples, making sure that we can deliver reliable, high quality data. The basis of the QC depends on four internal controls that are spiked into all samples and external controls in every Olink Analysis.
Standard deviations for Incubation Control 1, Incubation Control 2 and the Detection Control are calculated and should be below the predetermined threshold: 0.2 NPX, for the entire 96-well sample plate.
A sample plate median value is calculated for the Incubation Control 2 and the Detection Control, respectively. For each sample, the result for each of these internal controls is allowed to deviate no more than Â±0.3NPX from the plate median. If any or both of the internal controls exceed the 0.3 NPX limit, the sample will fail the QC. If more than 1/6th of the samples fail the QC, the run is deemed unreliable. The reason for the issues will then be evaluated and (if applicable) samples will be rerun.
The NPX Manager software displays the sample evaluation in two different views; the Plate View, showing NPX-values for each sample, and the QC view, showing deviation from the plate median in NPX units. In the example below, all samples but one, Sample 11 in well B2, pass the QC. For that sample, the Incubation Control 2 and the Detection Control deviate about 0.7 NPX and 1 NPX from the plate median, respectively, and the sample will therefore fail the QC. The NPX values for this sample will be included in the data export file but indicated with red font – download an example results file here.
NPX Managerâ€™s Plate View: NPX values for all 4 internal controls for all samples as plated in a 96-well plate. The Extension Control is used when calculating NPX and should therefore be 0 in this view, please read more on how NPX is calculated here: How is the data pre-processed? Sample well B2 indicates a sample with deviating internal controls and thus did not pass the quality control.
NPX Manager QC View: Samples are listed along the X-axis and deviation from plate median (in NPX) is shown on the Y-axis. For each sample a red and a blue dot are shown, these illustrate how much the Incubation Control 2 and Detection control, respectively, deviate from the plate median for that sample. The dotted red lines show the limits of +0.3 NPX and -0.3 NPX deviation from the plate median. The name of a sample that does not pass the QC is indicated in red font.