Olink NPX Signature Data Analysis Software User Manual

User Manual for Olink models including: NPX Signature, Data Analysis Software, NPX Signature Data Analysis Software, Analysis Software, Software

File Info : application/pdf, 48 Pages, 4.84MB

olink-npx-signature-user-manual-1

®
Olink® NPX Signature
User Manual
v1.0

Table of contents
1. Introduction.......................................................................................................................................................... 3 1.1 About this Manual ................................................................................................................................. 3 1.2 Intended Use and Intended Target Group ................................................................................. 3 1.3 List of Abbreviations ............................................................................................................................. 3 1.4 Associated Documentation ............................................................................................................... 3 1.5 About the Software ............................................................................................................................... 3
2. Computer Requirements ................................................................................................................................4
3. Install the NPX Signature Software on Windows ...............................................................................5 3.1 Uninstall the NPX Signature Software .........................................................................................5
4. Olink NPX Signature Study File (.NPX) ...................................................................................................6
5. Files and Information Needed for Analysis ............................................................................................ 7
6. Use the Olink NPX Signature Software ...................................................................................................8
7. Settings...................................................................................................................................................................9
8. Tutorials and In-Line Help .......................................................................................................................... 10
9. Plugins..................................................................................................................................................................11 9.1 Install a plugin .......................................................................................................................................11 9.2 Remove a plugin ...................................................................................................................................11
10. Quality control ...................................................................................................................................................12 10.1 Sample QC ...............................................................................................................................................12 10.2 Run QC ......................................................................................................................................................13 10.3 Assay QC ..................................................................................................................................................14
11. Normalization methods ................................................................................................................................15 11.1 IPC normalization ................................................................................................................................15 11.2 Intensity normalization v.2 ..............................................................................................................15 11.3 Intensity normalization .....................................................................................................................15 11.4 Calibrator normalization ..................................................................................................................16
12. Olink® NPX Signature - Operation ..........................................................................................................19 12.1 Import run data ....................................................................................................................................20 12.2 Enter study information ...................................................................................................................20 12.3 Verify plate layout and sample annotation ............................................................................20 12.4 Perform quality controls ...................................................................................................................21 12.5 Additional quality assessment ...................................................................................................... 24 12.6 Export data ............................................................................................................................................ 24 12.7 Create a Certificate of Analysis ..................................................................................................... 28
13. Troubleshooting.............................................................................................................................................. 29 13.1 Warning messages in Olink® NPX Signature ......................................................................... 29 13.2 Deviating controls for Olink Target in Olink® NPX Signature ........................................ 30 13.3 Sample flagged by Detection Control ........................................................................................33 13.4 Inconsistent results detected in Olink Target using Olink® NPX Signature ............. 38
14. Revision history ...............................................................................................................................................46
15. Appendix A:Related Documents .............................................................................................................. 47
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1. Introduction
1.1 About this Manual
Olink® NPX Signature User Manual provides the instructions needed for data processing when running Olink® Target 48, Olink® Target 96 panels. Specific instructions for Olink Target 48 are highlighted in the manual: Olink Target 48. This manual contains step-by-step instructions for analysis in Olink NPX Signature and software troubleshooting.
1.2 Intended Use and Intended Target Group
Olink NPX Signature is a data analysis software that is designed for the OlinkTarget analysis platform, including Olink Target 48, Olink Target 96, as well as for Olink Focus panels. It allows for importing data, validating data quality, and normalizing Olink data for subsequent statistical analysis. Olink NPX Signature is intended for research use only. Not for use in diagnostic procedures. All trademarks and copyrights contained in this material are the property of Olink Proteomics AB unless otherwise stated. For questions, guidance, and support, including questions regarding Focus panels, contact Olink Support at support@olink.com. Olink NPX Signature is intended to be used by trained users of Olink Target analysis platform
1.3 List of Abbreviations
%CV Coefficient of Variance CoA Certificate of Analysis IFC Integrated Fluidic Circuit (also called "chip" in this document) IPC Inter-Plate Control LOD Limit of Detection NPX Normalized Protein eXpression PCR Polymerase Chain Reaction QC Quality Control
1.4 Associated Documentation
All relevant Olink documentation is available from the Olink website www.olink.com/downloads.
1.5 About the Software
Olink NPX Signature is a standalone Windows software application that lets you import data from Olink Signature Q100, validate its quality, normalize it, and perform statistical analysis. The data is not required to be annotated or processed before importing it into Olink NPX Signature. The software can also process Biomark data.
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2. Computer Requirements

Components Operating System Processor Memory Disk Space

Minimum Windows® 10 or higher Intel® CoreTM i5 8 GB RAM 500 MB

Recommended
Intel® CoreTM i7 or higher 16 GB RAM or more 500 MB

3. Install the NPX Signature Software on Windows
The installer performs a first-time installation or an update to the current installation if it is already installed on your local computer. Any updates to the software retain all existing user settings and custom data. 1. Download the Olink NPX Signature Software to your computer from https://www.olink.com/. 2. Click the installer Olink NPX Signature Setup.exe. Olink NPX Signature is displayed in the Windows Start
menu for future use, and the following icon is displayed on your desktop:
To open the application, double-click the icon. IMPORTANT! Before using Olink NPX Signature for the first time or upon installation of an updated version, you are required to accept the end-user license agreement.
3.1 Uninstall the NPX Signature Software
If you need to uninstall Olink NPX Signature, it can be uninstalled using the Windows Add or Remove Programs application. 1. In the Control Panel, click Programs and Programs and Features. 2. In the Uninstall or change a program window, navigate to Olink NPX Signature, select it, and select
Uninstall. The application is uninstalled. All user data is retained where it was saved before the application was uninstalled, and your custom Olink NPX Signature settings are retained on the system.
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4. Olink® NPX Signature Study File (.NPX)
IMPORTANT! To enable NPX Signature's remote data functions to Signature Q100, ensure that the instrument is connected to the network and TCP port 8085 (enabled by default) is not blocked. Olink NPX Signature stores all study data in a file with the extension .NPX. This file is the single repository for all study-related data required by Olink NPX Signature to display, review, and export data for a study. Place this NPX file on your local computer or in a folder on the network. NPX files are typical ~510 MB in size but can be larger if you save the run data in the .npx file.
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5. Files and Information Needed for Analysis
Q100 Run Data. This is the Signature Q100 run data that can be imported into Olink NPX Signature without the need for annotation or processing in advance. The file types and information accessible for analysis are: · Olink Signature Q100 export file (.q100 or .zip) or · Olink Signature Q100 instrument with enabled remote connectivity or · Olink NPX Manager project file (.oaf) or · Fluidigm Real-time PCR exported heatmap or table file (.csv) See the tutorials and inline help in Olink NPX Signature for more information. Plate manifest file. This is the MS Excel file (.xlsx) needed to define the sample plate layout. Datafile version. This is the datafile version provided on the insert that is delivered with the Reagent kit. NOTE: The size of the .zip file that is transferred via USB from Signature Q100 might be larger than 20 MB. An NPX Signature study that includes run data in the .npx archive might be too large to email to Tech Support. If possible, email only the analysis data with the .npx extension.
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6. Use the Olink® NPX

Signature Software

You are required to accept the end-user license agreement the first time you install Olink NPX Signature or before using an updated version for the first time.

Click Olink NPX Signature in the Windows Start menu to see the top-level view:

Expand the main menu.

The expanded main menu.

Home. Access the main level of the application, where you see your most recently opened studies. Learn. Access all the integrated tutorials. See section 8. Tutorials and In-Line Help for more information. Create Study. Create a new study. Open Study. Open an existing study. Instruments. Show a list of available Olink Signature Q100 instruments to the Olink NPX Signature software over the network to import run data. Settings. Access the application settings. See section 7. Settings for more information. About. View the installed version of the application, copyright dates, and Open Source license information.
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7. Settings
Click Settings on the main menu to customize the view. General · Change the color theme of the application and charts. · Enable or disable the advanced user features. · Customize the certificate of analysis by adding a company name and logo. Layout Templates. - Review or create new sample plate layouts to define the sample types for each well. Panels. · Review all data files in the application. · Import or remove custom data files.
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8. Tutorials and In-Line Help
Olink NPX Signature contains browsable, integrated tutorials and in-line help.
Tutorials. Click Learn on the main menu to access integrated tutorials meant to assist you in learning the various features of Olink NPX Signature. Click a tutorial and navigate through the pages using Next or Previous.

You can also launch a tutorial by clicking

if you see it at the end of the in-line help content.

In-Line Help. Click the icon in the upper-right corner of a view to access the in-line help, which contains information about the usage of the current view. If the contextual help is longer than the allowed space, a scroll bar appears that lets you read the full text.

The following example shows a tutorial link to the current subject:

9. Plugins
Custom functionality may be added through a plugin library. Plugin libraries are delivered as .zip files and must be uncompressed into the user plugins folder before launching the software.
9.1 Install a plugin
1. Close all instances of Olink NPX Signature. 2. In Windows File Explorer, navigate to the following location:
%appdata%\olink-npx-signature\UserData\Plugins 3. In the Plugins folder, unzip the entire plugin .zip to a subfolder in the Plugins folder so that all the
contents of the zip are contained in their folder. For example, extract the MyPlugin.zip into the following folder: %appdata%\olink-npx-signature\UserData\Plugins\MyPlugin) 4. Open Olink NPX Signature. 5. Locate the plugin for a particular study in the Analysis or Report views.
9.2 Remove a plugin
1. Close all instances of Olink NPX Signature. 2. In Windows File Explorer, navigate to the following location:
%appdata%\olink-npx-signature\UserData\Plugins 3. Delete the folder for the plugin you wish to remove.
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10. Quality control
Olink has built-in quality controls in all multiplex panels. Each 92-plex panel contains 96 assays. Four of these are internal controls that allow for an in-process quality control designed to monitor different steps of the protocol: immuno reaction, extension and amplification/detection. The controls included are illustrated in Figure 1.
Figure 1. Outline of Olink internal controls for Olink® Target 96 panels.
· The two Incubation Controls consist of two different non-human antigens measured with PEA assays: Incubation Control 1 and 2. These controls monitor potential variation in all three steps of the reaction.
· The Extension Control is an antibody coupled to both DNA-tags (hence always in proximity). This controlmonitors the extension/pre-amplification and amplification/detection step and is used for normalization of the data.
· The Detection Control is a complete double stranded DNAamplicon which does not require anyproximity binding or extension step. This control monitors the amplification/detection step.
The internal controls are used for both sample and run QC as described below. The quality control of data is performed separately for each sample plate.
Olink® Target 48
Each Olink® Target 48 panel contains 48 assays, three of which are internal controls. These are: Incubation Control, Extension Control and Detection Control. Only one Incubation Control is usedin Olink® Target 48 panels, Incubation Control 1. The controls are used for sample and run QC as described below.
10.1 Sample QC
Each of the internal controls is spiked into all samples at a set concentration. The signals for these are therefore expected to be the same over the entire plate. Sample QC is performed using the Detection Control and Incubation Control 2. Within each run, the levels of these controls are monitored for each sample and compared against the plate median of all samples. If either of the controls deviate more than the acceptance criteria allow (see below), the sample is flagged. The Extension Control is used in the normalization step and in generation of NPX, and hence is not included in the quality control of data.
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Acceptance criteria for passing a sample: · Incubation Control 2 and Detection Control deviates < +/- 0.3 NPX from the plate median. Deviating values for the internal controls can be caused by, for example, errors in pipetting or pre-analytical factors in the samples that affect the performance of the controls. For more information on troubleshooting flagged samples, see section 8.3 Deviating controls for Olink Target in Olink® NPX Signature.
Olink® Target 48
Incubation Control and Detection Control are used for the sample QC of the Olink® Target 48 panels. The acceptance criteria for passing a sample are that Incubation Control and Detection Control deviate no more than +/-0.3 NPX from the plate median.
10.2 Run QC
The internal controls are also used in the run QC. This QC assesses the variation over the plate for each of the Incubation Controls 1 and 2 and the Detection Control. If the variation for one of the controls is too large (see below) the entire run is considered unreliable. Acceptance criteria for passing a run: · Standard deviation of Incubation Control 1 and 2 and Detection Control: < 0.2 NPX · Number of flagged samples: 1/6 of the total number of samples (i.e. 15 in a full plate)
Olink® Target 48
· Standard deviation of Incubation Control and Detection Control in samples: < 0.2 NPX · Standard deviation of Incubation Control and Detection Control in controls: < 0.5 NPX · Number of flagged samples: 1/6 of the total number of samples (i.e. 8 in a full plate) · Number of failed assays: 3 out of 45 (see section 10.3 Assay QC) · 1/3 of the Sample Control or Calibrator replicates are outside of LOQ for < 5 assays · Number of flagged Sample Controls: 1 · Number of flagged Calibrators: 1
If a too large variation is observed for either of the controls, go to the Plate QC -> Assay QC tab to evaluate the data. For example, if individual samples show extreme values or if a certain sample column is affected, these samples can be marked as failed, and the QC redone and re-evaluated. For more information on troubleshooting of this step, see section 13.2 Deviating controls for Olink Target in Olink® NPX Signature. In addition to passing or failing individual plates, ensure that no systematic bias is present in the data. The Plate metrics view in the bottom left corner alerts you to such issues. %CV is calculated using control samples if present in duplicates on each sample plate. The reported %CV is the mean %CV over all assays, and this is only calculated using data over LOD. A high %CV does not fail a run automatically but should be a cause for further investigation. · Reference value for Inter %CV: < 25% · Reference value for Intra %CV: < 15%
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Olink® Target 48
%CV is calculated using the Sample Control present in triplicates on each sample plate. The reported %CV is the mean %CV over all assays, and this is only calculated using data in pg/mL from non-flaggedreplicates within LOQ. A high %CV does not fail a run automatically but should be a cause for further investigation. · Reference value for Inter %CV: < 25 · Reference value for Intra %CV: < 15%
10.3 Assay QC
Olink® Target 48
Quality control of assays is performed for Olink® Target 48. An expected concentration value for the Sample Control for each assay is defined during the validation of the panels. The accuracy and precision for the calculated concentration values are evaluated for each assay. Acceptance criteria for passing an assay: · The accuracy of the calculated concentration mean for the Sample Control and Calibrator
for each assay must fall within +/-30% of the expected concentration. If this criterion is not met, the assay isnoted as "No Data" in the results file. · The precision of the calculated concentration for the Sample Control must be <30% for intra-CV. If the criterion is not met, the assay is noted as "No Data" in the results file. · A maximum of one of the Calibrator replicates and/or the Sample Control replicates can fall outside of the limits of quantification (LOQ). If the criterion is not met, the assay is noted as "No Data" in the results file. · The precision of the calculated concentration for the Calibrator is evaluated and should be <30% forIntra-CV. If the criterion is not met, the assay is noted as "Warning" in the results file.
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11. Normalization methods
Olink® NPX Signature supports three methods for between-plate normalization. Between-plate normalization adjusts median NPX levels based either on customer samples, IPC controls or Calibrators. Calibrator normalization is used for Olink® Target 48, see section 11.4 Calibrator normalization. An important concept when deciding on the normalization procedure for Olink® Target 96 panels is randomization, which in this context applies to the sample placement across the plates. For randomized studies with more than one plate, sample intensity normalization is the default normalization. For other studies, IPC normalization is the default. (See Randomization FAQ on the Olink website.)
Olink uses an arbitrary, relative quantification unit called Normalized Protein Expression (NPX). In qPCR, the x-axis value of the point where the reaction curve intersects the threshold line is called the Ct, or "Cycle threshold." This indicates the number of cycles needed for the signal to surpass the fluorescent signal threshold line. NPX is derived from the Ct values obtained from the qPCR using the following equations:
Extension Control: CtAnalyte CtExtension Control = dCtAnalyte
Inter-plate Control: dCtAnalyte dCtInter-plate Control = ddCtAnalyte
Adjustment against a correction factor: Correction factor ddCtAnalyte = NPXAnalyte
The correction factor is calculated by Olink during the validation of the panels.
11.1 IPC normalization
Three inter-plate controls (IPC) are included on each plate and run as normal samples. The inter-plate control is a pool of 92 antibodies, each with one of the pairs of unique DNA tags on it positioned in fixed proximity (i.e. 92 Extension Controls). The median of the three IPCs is subtracted for each assay, and this reduces variation between plates. This method is completely independent of the samples included on the plate and is therefore recommended for studys where complete randomization of samples cannot be guaranteed.
11.2 Intensity normalization v.2
The Intensity normalization v.2 adjusts the data so that the median NPX for a protein on each plate is equal to the overall median. Each plate is adjusted so that the median of all assays is the same on all plates. This method relies on the assumption that the true median of each plate is the same. One way of ensuring this is to randomize the samples beforehand. If there is total randomization, this method outperforms other normalization methods. If there are specific types of samples that are only available on certain plates, this normalization method should not be used.
NOTE: Only samples are included in this normalization. Negative Controls, IPCs and control samples are excluded. Compare to section "11.3 Intensity normalization".
11.3 Intensity normalization
Intensity normalization has been replaced by Intensity normalization v.2 as standard. The function remains as a tool for troubleshooting older datasets using this normalization method. This method uses the median of all samples on the plate, including Sample Controls, Negative Controls and IPCs.
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11.4 Calibrator normalization

Olink® Target 48

Calibrator normalization is used for Olink® Target 48 panels. A Calibrator is used to determine the protein concentration in the panel. The calibrator consists of pooled plasma from healthy individuals, with a spike-in of recombinant antigen for assays below LOQ. Randomization of samples is recommended but not mandatory.

For this panel, the Extension Control, the Calibrator and the bridging factor are used to calculate the normalization. NPX is derived from the Ct values obtained from the qPCR using the following equations for Olink® Target 48 panels:

Each sample is normalized with the Extension Control:

Ct Ct = dCt Sample i, Assay j

Sample i, Assay Ext ctrl

Sample i,Assay j

Each plate is normalized with its Calibrators to generate NPX levels and is adjusted against a pre-

determined bridging factor.

Bridging

factor

Assay

(dCtSample

Assay

Median(Calibrator

A Sample

Assay

))
j

NPXSample

i,Assay

NOTE: The NPX value will differ between the same protein measured on Olink® Target 96 and Olink® Target 48. This is mainly due to the fact that Olink® Target 48 uses a bridging factor and Olink® Target 96 a correction factor, and the sample used for plate normalization differs.

The NPX unit is unique to each protein assay, meaning that even if two different proteins have the same NPX values, their concentrations in pg/mL may differ.

The bridging factor is determined by Olink during the validation of the panels, and for each new kit lot, a lot-specific bridging factor is defined.

11.4.1 Results in standard unit (pg/mL)
The method for quantification of data in standard units (pg/mL) for Olink® Target 48 is described in this section. Method overview The method requires fewer sample wells in each plate/run for controls, allowing more wells to be used for customer samples. Before the run (during product development at Olink) 1. A precise pre-defined standard curve is established for each protein. 2. The triplicate Calibrator is used to adjust the standard curve along the y-axis to normalize
betweenplates and a second normalization, utilizing a bridging factor, is applied to ensure accuracy between production batches. 3. A four parameter logistic (4PL) model fit is performed to define the standard curve mathematicallywithin the measurement range for each protein in the panel. After each run 1. The median value of the Calibrator triplicates is used to adjust the measured samples along the y-axis (NPX) to normalize between plates. In other words, the samples are normalized in the same way as the standard curve is during development. The samples are also adjusted against the bridging factor, see formula above. 2. The adjusted standard curve model, is used to convert the measured NPX value to the protein concentration in pg/mL.
Standard curve During development, a thorough fine-tuned 32-point standard curve is developed for each protein biomarker simultaneously, including all 45 proteins. Multichannel pipetting and numerous replicates of the curves are used to minimize errors and establish an accurate immunoassay curve fitting. Due to the wide dynamic range of the PEA assays in the panel, a large spread of measurement points is required to cover the
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entire range. Eight measurement points, which are commonly used in many technologies, would not suffice for Olink's multiplex measurement of 45 assays. The predefined standard curves avoid operator- dependent reconstitution and pipetting of standard curves at each lab and for each run. The figure below shows an example of a point standard curve defined for one assay during development. Standard curves for each assay can be found via the panel product page (www.olink.com/ t48cyt).
Figure 2. A 32-point standard curve defined for each assay during development.
4PL model A 4PL model curve fit is used to describe the immunoassay standard curve, indicated by the turquoise line in Figure 2. When running a study, the measured patient sample value (represented by the dotted blue arrows in Figure 2) is related back to the adjusted standard curve model which translates the measured value to the protein concentration in pg/mL using the equation below. Where A= asymptote (a constant value) at low doses, B= slope factor, C= EC50 (mid-range concentration) and D= asymptote at high doses. These parameters are indicated in Figure 3 . An example of a point standard curve defined for each assay during development..
Figure 3. An example of a point standard curve defined for each assay during development.
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Repeated testing and validation show that the 4PL curve fitting describes the standard curve well and can be used to correctly estimate the protein concentration in analyzed samples within the limits of quantification. The approximate lower and upper limits of quantification (ULOQ and LLOQ) are defined during the development of the panel, and specifications for LLOQ and ULOQ are established for each lot. See the panel specific Validation data document available on the Olink website (www.olink.com) for more details. Results reported in pg/mL Figure 4 and the list below provide an overview of how data in pg/mL is reported in the results file. · Data between LQL and ULOQ is reported as a pg/mL value and shown in the white areas of the graphs. NOTE: Lowest Quantifiable Level (LQL) is defined as the value used as the lower limit, LLOQ (default) or plate LOD (when plate LOD > LLOQ). · Data below the lowest fitting parameter in the 4PL curve fit model cannot be calculated and is indicated
as NaN in the red areas of the graphs. · Data >ULOQ is indicated as >ULOQ in the red areas of the graphs. Values above ULOQ are not reportedin
pg/mL due to high risk of misinterpreting hooking data. · Data below LQL is presented in pg/mL value in the red areas of the graphs. Values below LQL
should be treated with caution due to decreased precision and accuracy in the lower range and should not be usedfor individual comparison to reference values. For a more detailed list describing the results file, see section 12.6 Export data.
Figure 4. Overview of how data in pg/mL is reported in the results file.
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12. Olink® NPX Signature Operation
Input data for Olink® NPX Signature are run files from Q100 Signature, Ct values exported from the Fluidigm Real-Time PCR Analysis software, NPX Manager .oaf project files and NPX Signature .npx study files. After quality control and normalization against the Extension Control, the IPC, and a correction factor, the output data are obtained in Normalized Protein eXpression (NPX) values. NPX is an arbitrary unit on a log2 scale, where a high NPX value corresponds to high protein concentration.
Olink® Target 48
For Olink® Target 48, normalization against the Extension Control, Calibrator, and bridging factor is performed. The default output data is in standard concentration units (pg/mL). This section describes how you analyze data step-by-step in Olink® NPX Signature. Olink® Target 48-specific information is highlighted. The following steps are included in the standard operating procedure: 1. Select Create Study 2. Enter study information 3. Click Create 4. Import run data. 5. Verify panel and plate layout information 6. Under Details, enter optional study information 7. Select normalization method. 8. Perform quality controls including additional quality assessment. 9. Export data and certificate of Analysis under Report tab.
Figure 5. Olink® NPX Signature Study Overview.
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12.1 Import run data
Add run data to existing study Expand the main menu by clicking the three horizontal lines in the top left corner. Select Import Run Data. · To import run data directly from a Signature Q100 instrument, select Olink Q100 Connect. Select the
desired run(s) and press Next. Select Next again and verify panel and plate layout information. Click Add. The Study Overview will appear. · To import run data files from a local server, select Import Run Data, and click Run Data Files. Select files the desired run data file(s). Valid options are Olink Signature Q100 export files, .q100 or .zip, Olink NPX Manager study files, .oaf, Fluidigm Real-Time PCR exported heatmap or table files, .csv. NOTE: A study cannot include different panel types at the same time. It can consist of only runs from Olink® Target 48 panels, only runs from Olink® Target 96 panels or, only runs from Olink® Focus panels.
During import, Olink® NPX Signature alerts if duplicate sample names are found since unique sample and assay names are necessary for proper handling of the data. Change sample names if needed. Sample names can be changed directly in Olink® NPX Signature by importing a manifest in the Plate Manifests view. For more information see built-in help in the Plate Manifests view.
12.2 Enter study information
Study information can be entered in the Study -> Details view · Enter information in some or all the following fields to display it in the CoA:
Sample Type Customer Information Business Development Manager Lab Info Report Notes QC Notes for CoA Internal Notes NOTE: Any entered Notes will be saved in the Olink® NPX Signature study (.npx) file.
12.3 Verify plate layout and sample annotation
1. Select Plate QC-> Plate Layout to display the imported runs for each Olink panel and plate. 2. Under Plate Details, verify that the correct data file version is selected in the drop-down menu.The same
data file version will be used for all runs of the same Olink panel in the study. 3. Verify that the plate layout is correct. To change the plate layout, select one or several wells and right-click
to change the well type. Available types are: · Sample (S) · Control (C) · Negative Control (Neg) · Inter-plate Control (IPC) · N/A
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Olink® Target 48
The following additional well types are available for Olink® Target 48: · Control (C) · Calibrator (Cal) Calibrator, Negative Control, and Control for Sample Control must be annotated for calibration normalization and QC to be performed. Up to four different control sample types (Control 1 4) can be selected, but only Control 1 should be used for Olink® Target 48 Cytokine.
4. Repeat steps 2 and 3 for all imported plates before continuing.

12.4 Perform quality controls
1. Perform the QC for each plate separately using the Sample QC tab.
2. Perform quality assessment for all plates using the other views as described in section 7.5 and then make an overall assessment.
The acceptance criteria for passing QC are described in Table 1. Quality Control guidelines for Olink® Target 96. and Table 2. Quality Control guidelines for Olink® Target 48..

Table 1. Quality Control guidelines for Olink® Target 96.

Criteria Run QC Std. Dev. of Incubation Control 1 Std. Dev. of Incubation Control 2 Std. Dev. of Detection Control Number of flagged samples
Sample QC Incubation Control 2 deviation Detection Control deviation

Recommended value
< 0.2 NPX < 0.2 NPX < 0.2 NPX 1/6 of total number of samples on plate (15 samples on a full plate)
< ±0.3 NPX from plate median < ±0.3 NPX from plate median

Table 2. Quality Control guidelines for Olink® Target 48.
Olink® Target 48
Criteria Run QC Controls Std. Dev. of Incubation Control Samples Std. Dev. of Incubation Control Controls Std. Dev. of Detection Control Samples Std. Dev. of Detection Control Number of flagged samples
Number of failed assays Calibrator or Control replicates outside LOQ
Number of flagged Sample Controls Number of flagged Calibrators Sample QC Incubation Control deviation Detection Control deviation Assay QC Accuracy Sample Control Precision Sample Control Calibrator or Control replicates outside LOQ
Precision Calibrator

Recommended value
< 0.5 NPX < 0.2 NPX < 0.5 NPX < 0.2 NPX 1/6 of total number of samples on plate (8 samples on a full plate) 3 out of 45 1/3 of the Sample Control- or Calibrator replicates are outside of LOQ for < 5 assays 1 1
< ±0.3 NPX from plate median < ±0.3 NPX from plate median
+/-30% of the expected concentration Intra-CV <30% 1/3 of the Sample Control- and/or Calibrator replicates are outside of LOQ Intra- CV <30%

Tip: If Std. Dev. is higher than the recommended value, evaluate the QC graph below to identify any extreme outlier samples. Exclude these samples from the QC analysis (click on the sample in the graph and select QC analysis: Excluded) and re-evaluate the run QC again.

12.4.1 Perform a quality assessment
Parameters that need to be evaluated for each run are listed in Table 3. Quality assessment for Olink® Target 96..

Table 3. Quality assessment for Olink® Target 96.

Olink® Target 96:
Information Assay LOD

Recommended value |LOD| < 2.5 NPX
10 (number of assays) > 10 (number of assays)

Comment
If |LOD| > 2.5 please contact Olink Support for guidance. Plate ANOVA
No warning.
May be caused by insufficient randomization or other issues.
NOTE: These guidelines are based on a two-plate study. The larger a study is, the smaller the detectable differences become.

Row/Column
%CV Intra %CV Inter Detectability

ANOVA < 30 (number of assays) 30 60 (number of assays)
60 90 (number of assays)
< 15% < 25% > 75% of the samples

No warning.
Possible randomization issue. See section 8.5 Inconsistent results detected in Olink Target using Olink® NPX Signature.
Re-run plate or otherwise ensure that this is not the result of a poorly executed run.
For high values, investigate the Assay QC view.
See section 13.4 on page 38 and section 13.2 on page 30.
See section 13.4 on page 38 and section 13.2 on page 30.
See document on https://www. olink.com/members-site/.

If all plates pass quality control and assessment, the data analysis is finished, and the data is ready for export and reporting.

Table 4. Quality assessment for Olink® Target 48.

Olink® Target 48

Information Plate ANOVA
Row/Column ANOVA

Recommended value Comment

10 (number of assays) No warning.

> 10 (number of assays) May be caused by insufficient randomization or other issues. If samples were not randomized, this warning can bedisregarded.

NOTE: These guidelines are based on a twoplate study. The larger a study is, the smaller the detectable differences become.

< 15 (number of assays)

No warning.

15 30 (number of assays)

Possible randomization issue. See section 8.5 Inconsistent results detected in Olink Target using Olink® NPX Signature

30 45 (number of assays)

Re-run plate or verify that this is not the result of a poorlyexecuted run.

For high values, investigate the Assay QC view.

%CV Intra

< 15%

See section 13.2 on page 30.

%CV Inter

< 25%, maximum 40% See section 13.2 on page 30.

Detectability

> 75% of the samples The limit for detectability is determined from observedvalues in plasma.

12.5 Additional quality assessment
12.5.1 Additional quality assessment Olink Target panels
NOTE: Switch between Ct and NPX in each view e.g. to evaluate the effect of normalization or to search for position effects in internal controls and assays.
Olink® Target 48
For Olink® Target 48, the quantified value setting can also be used to evaluate these effects.
12.5.2 Instructions
1. On the Assay QC tab, view the values of internal controls and assays (change assay in the drop-down menu above the plate layout) for each sample. For example, search for specific patterns that may indicate technical errors.
2. On the Table tab, evaluate patterns of data below LOD between sample plates in a study and/or remove specific assays or single datapoints from a study/sample plate.
3. On the Heatmap tab, search for outlier samples and/or visualize homogeneity of Negative Controls and IPCs (change sample type in the drop-down menu).
Olink® Target 48
· For these panels, the homogeneity of Calibrators and Sample Controls can also be visualized using the Heatmap tab. Use the Quantified Value setting to provide an overview of failed assays which will be marked in black.
On the Detectability tab, evaluate the detection limit for each assay and sample plate (optimal range for LOD ± 2.5 NPX). Assess if deviating LOD for one sample plate affects detectability on other sample plates.
Olink® Target 48
· For these panels, some assays can be expected to have LOD values above 2.5 and this should not affect the number of samples that can be quantified above the plate LOD. The Detectability view can be used to assess if such assays have a high number of samples outside LOQ.
On the Z-scores tab, display differences between sample plates or distributions within a plate.
12.6 Export data
In the Report -> Data tab, Data can be exported to a Microsoft Excel XLSX file or CSV file for use in other applications and for statistical analysis.
Olink® Target 48
Data can be exported both as NPX and pg/mL in separate reports, by selecting the desired Data type. Default export is pg/mL.
1. Select NPX Value on the status bar in the upper left corner of the main window.
Olink® Target 48
Select Data type: Quantified Values to export data in pg/mL.
2. Under Study Options on the Study tab, select the output option for NPX values below LOD. The default is "Actual Value", which displays the actual measured value for each data point.
24

Figure 6. Olink® NPX Signature Export Data view.

3. Figure 6 shows the Export Data view with the recommended default settings. These settings may be adjusted to change the format, layout, or to add additional data to the export. See Table 4 for more information.
4. In the Report -> Data tab, mark the desired runs to be included in the report and click Generate.
5. Enter a filename and location in the Save dialog, select an export format (XLXS files (Excel) or CSV files) and click the Save button.

Table 5. Data export options.
Setting Export format Layout
Export Samples Export Control Samples Export Plate Controls Export Assay Results Export Control Assay Results Export QC data
Export Plate LOD

Description Excel or CSV For CSV format, column separator and decimal separator can be configured. Wide format or Long format. Wide is the default matrix format and Long format will output one line per datapoint.
NOTE: In Excel, there is a number of rows limit of 1 048 576. If this number is exceeded, Excel export will not work, and CSV format should be selected instead. Export data for customer samples. Export Control samples. Export IPC, Plate Control, Negative Control or Calibrators. Export results for assays. Export results for control assays.
Export deviation from median for Incubation Control 2 and Detection Control.
Olink® Target 48
Deviation from median for Incubation Control and Detection Control is exported in NPX. Data for the controls cannot be calculated in pg/mL and will be blank in the results file for quantified values. Will export plate LOD for each plate as well as max LOD.

12.6.1 Considerations
· Samples that are flagged by the analysis are reported in red text. NPX-values that are below maximum plate LOD have a red background.
· Samples, assays, or data points that have been marked as failed in the software show empty cells in the data export.
Olink® Target 48
In the results file in standard units (pg/mL) the data is reported as follows: · Data between LQL and ULOQ is reported as pg/mL value in white cells. NOTE: Lowest Quantifiable Level (LQL) is defined as the value used as the lower limit, LLOQ (default) or plate LOD (when plate LOD > LLOQ) · Data >ULOQ is indicated as >ULOQ in red cells. · Data below LQL is presented in pg/mL value in red cells. · Data below lowest fitting parameter in the 4PL curve fit model cannot be calculated and is
indicated as NaN in red cells. · Failed data points (either because of assay failure, sample failure or chip failure) are indicated as · No data in grey cells. · For samples and assays with QC warning, values are indicated as described above but marked in
red text. Data from samples and assays that do not pass QC should be treated with caution. · Failed data points (either because of assay failure, or sample failure) are indicated as actual
values in grey cells. · Failed data points (because of chip failure) are indicated as No data in grey cells. · For each plate and assay, values for LQL, LOD, LLOQ and ULOQ, as well as results for assay QC in · Assay warning, are presented on separate rows below the data for the samples. · Missing data frequency is reported for each assay and indicates the percentage of samples with
values <LQL, >ULOQ as well as failed data.

Table 6. Overview presentation of data in standard units (pg /mL) in results report.

Result and QC results

In Data report

Sample QC
Pass Fail Pass Pass Pass Pass Pass Pass

Calibrator QC precision
Pass Pass

Sample Control QC precision and/ or accuracy Pass Pass

Fail

Pass

Fail

Pass

Calibrator and/or Sample Control QC replicates within LOQ Pass Pass
Pass
Pass Fail Pass
Pass
Pass

Data between LQL and ULOQ
Yes Yes
Yes
Yes Yes No; < curve fitting No; < LQL
No;

Data Output Format
pg/mL pg/mL
pg/mL
pg/mL pg/mL NaN
pg/mL
> ULOQ

Sample Assay QC Notes QC

Pass Pass Warning Pass
Pass Warning
Pass Fail Pass Fail Pass Pass

Treat with caution
Treat with caution

Pass Pass Pass Pass

Treat with caution

In the NPX results Excel file that can be optionally exported, data will be presented as follows:

· As NPX values. · Values below maximum plate LOD have a red cell background. · Data for samples with a QC warning are indicated in red text. Data from samples that do not pass
QCshould be treated with caution. · The maximum plate LOD value for each assay is presented on a separate row below the data for thesamples
and is indicated as LOD. · Missing data frequency is presented for each assay and indicates the percentage of samples with values
below Maximum plate LOD.

The layout is described in Table 7. Parameters in brackets represent quantified data.

Table 7. Layout of exported NPX data.

Study Name

Olink® NPX Signature version

NPX data or Quantifed data

Panel

Panel 1

...

Panel x

Panel 1 ... Panel x Panel 1 ... Panel x

Assay

1 2 3 ... 96

Uniprot ID Olink ID Sample #1

1 2 3 ... 96 1 2 3 ... 96 1 2 3 ... 96

Sample #2

1 2 3 ... 96

Sample #x

1 2 3 ... 96

... [Assay warning]

1 2 3 ... 96

[Lowest quantifiable 1 2 level]

Max LOD [Plate

LOD]

[ULOQ]

[LLOQ]

Missing Data freq. 1 2 (%)

Normalization type 1 2

3 ... 96
3 ... 96
3 ... 96 3 ... 96 3 ... 96
3 ... 96

1 2 3 ... 96 Plate ID
1 2 3 ... 96 1 2 3 ... 96 1 2 3 ... 96 plate_1.
csv 1 2 3 ... 96 plate_1.
csv 1 2 3 ... 96 plate_1.
csv

Plate ID
plate_x. csv plate_x. csv ... plate_x. csv

QC Warning
Pass / Warning Pass / Warning Pass / Warning

1 2 3 ... 96 plate_1. csv
1 2 3 ... 96 plate_1. csv
1 2 3 ... 96 [plate_1. csv]
1 2 3 ... 96 1 2 3 ... 96 1 2 3 ... 96
1 2 3 ... 96

plate_1. csv
plate_1. csv
[plate_1. csv]

Table 8. Specification for column content in the NPX file.

Column

Description

SampleID

The annotated sample ID

Index

Well index in 96 plate

OlinkID

OlinkID for assay

UniProt

UniProt ID for assay

Assay

Gene name for assay

MissingFreq. Frequency of missing data (below LOD or NaN)

Panel

Panel name assay belongs to

Panel_Lot_Nr Lot number for the panel

PlateID

Name of the plate the sample was run on

QC_Warning Indicates whether the sample passed QC or not

LOD

LOD value for assay

NPX

NPX value

Normalization Type of normalization used in study

Assay_Warning Indicates whether the assay passed QC or not

Type String Integer String String String Float String Integer String String PASS, WARN or FAIL Float Float String Plate control or Intensity String PASS or WARN

12.7 Create a Certificate of Analysis
Once all data has been thoroughly checked, Olink® NPX Signature can generate a Certificate of Analysis (CoA) for the study. The CoA includes information and QC parameters for the study.
Go to Report -> Certificate and select all panels to be included in the CoA. Click Create to save the CoA as a PDF document. A preview of the document will appear.

13. Troubleshooting
This chapter describes issues that may arise during use of Olink® NPX Signature, or data issues during analysis, and how to solve these issues.
13.1 Warning messages in Olink® NPX Signature
The following warnings can be displayed: 1. Opening Study
a. Could not identify the panel The panel datafile associated to plates in the study cannot be located and thus not identified.
Navigate to Settings -> Panels and import the required panel data file. 2. Importing Run Data
a. Quality Threshold does not meet specified criteria 0.5. The Quality Threshold defined for analysis was not set to 0.5. Open Run data in Fluidigm Real-Time
PCR and export the data after analyzing with a quality threshold of 0.5. b. Baseline Correction method is not set to Linear. The Baseline Correction method defined for analysis was not set to Linear. Open run data in
Fluidigm Real-Time PCR and export the data after analyzing with a baseline correction method of Linear. c. Unsupported plate format The run data was not acquired for a supported IFC. d. Unsupported probe-type count, expected 1. The run data was not acquired with a supported protocol e. Panel types must match in study The study already contains a plate for one-panel type (Olink 96, Olink 48, etc.). All plates in the study must be run for the same panel type. f. Run barcode already exists in a study for the panel The run data for this IFC has already been imported to the study. g. Data integrity file not found in the archive The run data checksum file was not found in the Q100 Signature export. The run data must be analyzed and exported from Real-Time PCR before importing. h. Could not locate data file in file The run data analysis export was not located in the Q100 Signature export file. This may be due to a corner finding error during analysis, due to: o Insufficient amount of ROX dye in corner wells o Contrast not optimal o Edge sample or assay is not loaded (e.g. air bubbles or particle clogging of the IFC) Unzip the run data archive and open the ChipRun.bml in Fluidigm Real-Time PCR to analyze and export data. Manually set the corners using Fluidigm Real-Time PCR Analysis Software, export in .csv format and import in NPX Signature
29

Make sure to zoom in to clearly see where the wells should be (if empty). All wells will not fit optimally, the important ones are the four corner wells.
i. Datafile checksum not verified The run data contained in the Q100 Signature export file failed to validate using the checksum
values generated when the analysis was performed. This may indicate that the run data has been modified after it was initially analyzed when the run was going to be completed Unzip the run data archive and import the results_all.csv, results_all.csvx, or results_heatmap.csv file directly. 3. Importing Panel Datafiles a. Data file already exists A panel definition with the same name and version is already imported in the application. 4. Importing Sample Manifest Files a. Unrecognized file extension for sample plate manifest The file format could not be processed. Supported formats are Fluidigm Real-time PCR comma value separated plate definitions and NPX Signature XLSX format. b. No Sample-ID defined in input data The sample manifest import file does not define the Sample-ID column c. No Plate-ID defined in input data The sample manifest import file does not define the Plate-ID column

13.2 Deviating controls for Olink Target in Olink® NPX Signature
If the normalized value for internal controls or a specific sample deviates from the rest of the sample set, the sample is flagged. A flagged sample should not be excluded from the data export but may be excluded from the QC analysis. See section 12.4 Perform quality controls on page 21 for evaluation of the data.

Table 9. Flagged samples

Issue A sample is flagged

Explanation
A sample is flagged when one or several internal controls deviate for that specific sample.

Eason
All flagged samples in a study are shown under Study - > Summary in the table Flagged or failed samples.

Action
The behavior of the internal controls makes it possible to understand why the sample is flagged. See the rest of this section for more information. For more detailed information about flagged samples, click the Plate QC -> Sample QC tab (Figure 13) and the Assay QC tab (Figure 16).

Figure 7. The Sample QC tab, where the left circled sample is flagged by Incubation Control 2, and the right circled sample is flagged by the Detection Control. Both of these datapoints are outside the recommended threshold.
Figure 8. Assay QC tab for samples shown in Figure 7. The circled sample to the left has a lower NPX for Incubation Control 2. The circled sample to the right has a higher NPX for the Detection Control and lower NPX for the Extension Control.
31

13.2.1 Sample flagged by Incubation Controls

Issue

Explanation

Reason

Action

A sample is flagged by the Incubation Control.

The Incubation Controls deviate.

If the two Incubation See examples in Table Controls deviate, but 10. Common reasons not the Extension and for deviating Incubation Detection Controls, this Controls. is an indication that something (most likely in the sample) is affecting only the Incubation Controls.

Table 10. Common reasons for deviating Incubation Controls.

Possible cause Explanation

Solution

Sample matrix

If the sample matrix for the flagged sample is different from the others on the same plate, the incubation environment can be different. This can make the reaction slightly more or less efficient.

Do the QC evaluation for one sample type at a time.

Sample volume The sample volume is too high or too or concentration low.

Set the sample as failed in the Plate Layout tab. If applicable, run the sample again and make sure the correct volume is used. See Figure 7 on page 31, column 10 for example.

Sample quality

If the sample is stored in a freezer for long periods of time, it can evaporate. A more concentrated sample can increase possible matrix effects, and thus make the internal controls more or less efficient.

Sample type

Olink assays are validated for serum Standardize samples as much as possible. and plasma. Other sample matrices can (E.g.: for lysed cells and tissue, use similar contain factors that interfere with the concentration of protein in each sample) immuno reaction step.

13.3 Sample flagged by Detection Control

Issue

Explanation

Reason

Action

A sample is flagged by Detection Control.

The NPX value of the Detection Control for this specific sample deviates more than 0.3 NPX from the plate median.

One example can be This type of flag is

seen in Figure 7 on page generally caused by

31 and in Figure 8

the same reasons as

on page 31. Data is

flagged Incubation

normalized using the Controls. It is not

Extension Control. Since uncommon that both

both Incubation Controls the Incubation Control

and the Extension

2 and Detection Control

Control display

flag simultaneously, but

decreased NPX-values, with opposite "direction"

the normalization step in the Sample QC.

will adjust the data. The This is seen when the

Detection Control that flagged sample affects

did not show decreased both the extension and

NPX-values will be

immunoassay step, but

overcompensated

to different extent. See

by normalization

examples in Table 10.

against the Extension

Control and thereby

deviate from the rest

of the sample set after

normalization.

13.3.1 Deviating external controls

Issue

Explanation

Olink® Target 96: High Intra-CV value is larger

Intra- CV value

than 15%.

Reason
The control samples differ within the sample plate.

Action
Check sample annotation of the control samples and that they pass QC. Check the row pattern for rows A and B to make sure that none of the rows is an outlier (e.g. issue with a channel on one of the multichannel pipettes). Fail one or both of the control samples if remaining data on the plate pass all QC analysis and assessment.

Issue
Olink® Target 48
High Intra-CV value
Olink® Target 96:
High Inter-CV value
Olink® Target 48
High Inter-CV value

Explanation

Reason

Action

Intra-CV value is larger than 15%.

The Sample Control replicates differ within the sample plate.

Check sample annotation of the Sample Controls and that they pass QC. Check the row pattern for rows A, B and C to make sure that none of the rows is an outlier (e.g. issue with a channel on one of the multichannel pipettes). If one Sample Control replicate deviates from the other two and this pattern can be confirmed for several assays, this could indicate a manual issue with this replicate. In such case, that replicate can be failed and the remaining replicates used for CV calculation. Please contact support@ olink.com for further guidance

Inter-CV value is larger than 25%.

The control samples differ between sample plates.

Check sample annotation of the control samples and that they pass QC. If intensity normalization has been used, ensure that randomization assumption is valid by comparing with IPC normalization.

Inter-CV value is larger than 25%.

The Sample Control Check sample annotation

replicates differ

of the control samples and

between sample plates that they pass QC. If one

Sample Control replicate

deviates from the other

two and this pattern can

be confirmed for several

assays, this could indicate

a manual issue with this

replicate. In such case that

replicate can be failed and

the remaining used for CV

calculation. Please contact

support@ olink.com for

further guidance.

Issue
Olink® Target 96:
High LOD (> 2.5 NPX)

Explanation

Reason

Action

Background levels, · Sample and/or primer

values in Negative columns double

Controls, are

pipetted during

increased above loading of IFC

expected levels

· Rerun from Detection step

· Technical issue (increased background may occur sporadically due to random interaction of probes or airborne contamination). Seen for all three Negative Control wells for specific assays.

· If high LOD does not affect the detectability for the assay, continue data analysis
· If detectability is affected in a multi plate study, the affected assay can be excluded from the affected plate and data analysis continues for remaining plates or rerun from immuno reaction step

· Please contact support@ olink.com for further guidance

Olink® Target 96:
Large number of high and/or low LODs (outside ± 2.5 NPX)

Calculated LODs are out of expected range

· Sample/IPC contamination of Negative Control
· Sample and/or primer columns switched during loading of IFC

· If one well affected: Exclude contaminated Negative Control
· If two or more Negative Controls are affected: Rerun samples
· If noted during lab work, contact Olink support

· Wrong datafile · Wrong panel · Contamination of
Negative Controls · Wrong annotation of
negative controls or IPCs

· Confirm datafile; reanalyze data
· Please contact support@ olink.com for further guidance

Olink® Target 96
Large deviation in LOD between plates in study

LOD values differ more than 1 NPX between sample plates in the same study

· Contamination of one · Evaluate which plate and/

or several wells

or well is contributing to

· Pipetting error

the large deviation

· Technical issue

· Exclude affected well if

(increased

that can be defined

background may

· Exclude assay on affected

occur sporadically due plate or rerun from

to random interaction immuno reaction step

of probes). Seen for all · Please contact support@

three Negative Control olink.com for further

wells for specific

guidance

assays.

Olink® Target 48
Issue
High LODs that affect the number of samples that can be quantified above LOD.

Explanation

Reason

Calculated LODs are out of expected range

· Wrong datafile · Wrong panel · Contamination of
Negative Controls · Wrong annotation
of Negative Controls, Sample Controls or Calibrators.

Action
· Confirm annotation of Negative Controls, Sample Controls and Calibrators
· Confirm datafile; reanalyze data
· Please contact support@ olink.com for further guidance

Olink® Target 96:

Large deviation between NPX values for

triplicates of IPC and/or triplicate samples

Negative Controls on the deviate more than

same plate

0.5 NPX

Olink® Target 48

· Contamination of one or several wells
· Pipetting error

· If one well affected: Exclude that well
· Rerun if several wells are affected

Sample Control intra CV>30% and/or accuracy > +/- 30% causing many failed assays

Sample Control intra CV>30% and/ or accuracy > +/30%

Sample Control

Check sample annotation

replicates differ within of the Sample Controls and

the same plate

that they pass QC. Check

the row pattern for rows

A, B and C to make sure

that none of the rows is

an outlier (e.g. issue with

a channel on one of the

multichannel pipettes).

If one Sample Control

replicate deviates from the

other two and this pattern

can be confirmed for

several assays, this could

indicate a manual issue

with this replicate. In such

case that replicate can be

failed and the remaining

used for CV and accuracy

calculation. Please contact

support@ olink.com for

further guidance.

Olink® Target 48
Issue
Calibrator intra CV>30% resulting in many assays with warning

Explanation Calibrator CV>30%

Reason
Calibrator replicates differ within the same plate

Action
Check sample annotation of the Calibrators and that they pass QC. Check the row pattern for rows F, G and H to make sure that none of the rows is an outlier (e.g. issue with a channel on one of the multichannel pipettes). If one Calibrator replicate deviates from the other two and this pattern can be confirmed for several assays, this could indicate a manual issue with this replicate. In such case that replicate can be failed and the remaining used for CV calculation. Please contact support@ olink.com for further guidance.

13.3.2 Missing sample data

Issue

Explanation

Missing sample data in Data for some samples

exported results file

are not exported.

Reason

Action

This can be caused by not using unique sample names.

Ensure that sample names are unique by checking Plate QC -> Samples tab or change sample type to Control for non- unique samples to force export of data for these samples.

13.4 Inconsistent results detected in Olink Target using Olink® NPX Signature

13.4.1 Variation between plates - common reasons

Issue

Explanation

Reason

Action

The "Plate ANOVA" value in the Panel metrics box (Figure 9) shows a high value. The variation is also shown on the Plate QC -> Z-scores tab (Figure Figure 38. Plate variation that should be solved by intensity normalization.).

The total NPX varies between plates.

High Inter-CV value

Inter-CV value is larger than 25%.

May be caused by insufficient randomization, meaning that the distribution of samples from different groups is not the same for all plates in the study.
The control samples differ between sample plates.

If the samples are completely randomized, make sure that intensity normalization has been used. The results will then look like the example in Figure 11 on page 39, the same results as in Figure 10 on page 39 after intensity normalization.
Check sample annotation of the control samples and that they pass QC. If intensity normalization has been used, ensure that randomization assumption is valid by comparing with IPC normalization.

Figure 9. The Panel metrics field, with a Plate ANOVA value that indicates variation between plates.
38

Figure 10. Plate variation that should be solved by intensity normalization.
Figure 11. The same results as in Figure 38, after intensity normalization. 39

13.4.2 Variation within a plate - common reasons

Issue

Explanation

Reason

Action

The columns or row ANOVA in the Plate Metrics or Panel Metrics box show high values. The effects are visualized in the Assay QC tab.

Areas of a plate (rows The variation is caused

and/or columns) have by the sample position

systematically higher or on the plate. Within-

lower values (Ct and/or plate effects are often

NPX) than the rest of the caused by laboratory

plate.

mistakes.

See examples in Table 11.
NOTE: Make sure that the deviation cannot be explained with biological variation, i.e. that a unique sample group is placed on one column/row.

Position effects/patterns: visible only for Internal controls

All internal controls are affected on Ct-level in a similar manner (all increased or decreased)

One sample column or row consistently show deviating values

Pipetting error, e.g. pipette not preconditioned

Exclude data from analysis and/or rerun

Mistake during pipetting Refer to Figure 12 on

of the incubation

page 42.

solution, extension

solution, detection

solution or samples

Evaporation due to incomplete sealing of plate during incubation or PCR steps

Exclude data from analysis and/or rerun

Different sample matrices

QC matrices separately

Incubation Controls and Extension Control differ from Detection Control on Ct-level

Incubation and Extension Controls show the same pattern, Detection Control shows another pattern

PCR instrument not good at keeping 50°C, Detection Control not affected by extension step

Rerun from immuno reaction step

May be pre-analytical (interfering factors may affect all controls but the Detection Control)

Please contact support@olink. com for further guidance

Incubation Control/s differ from Extension and Detection Controls

One or both Incubation Controls show deviating pattern

Pre-analytical factors in samples that interfere with the immuno reaction

Few deviating samples: Exclude samples from subsequent statistical data analysis. > 1/6th of samples deviating: Contact support@olink.com for further guidance

Position effects/patterns: visible for samples and Internal controls

Columns/rows/parts of sample plate

Patterning that follows Different sample sample naming/sample matrices matrices

QC matrices separately

Issue
Gradient over the sample plate

Explanation
Upper or lower part or left and right part of the plate affected with clearly deviating values

Reason

Action

IFC issue Poor vortexing in extension step Poor mixing of detection mix Uneven temperature in PCR block

Rerun Contact support@olink. com for further guidance

Table 11. Common problems and reasons for within-plate effects.

Problem

Possible cause

Figure reference

First column different than the rest of the plate (observed for internal controls)

The pipette was not preconditioned.

A specific column or columns is/are different than for the rest of the plate (observed for internal controls)

A mistake when the incubation solution, extension solution, detection solution or samples were pipetted.

One row consistently different from the The pipette tip in that specific position rest of the plate (observed for internal was not tight enough. controls and/or samples)

A gradient from row A to row H

Pipetting was done at an angle. The plates were not sufficiently vortexed.

Deviating Z-score distribution for single assays on one plate

Primer contamination.

Refer to Figure 13 on page 43.
Refer to Figure 16 on page 44. Refer to Figure 15 on page 44

13.4.3 Extreme outliers - common reasons

Issue

Explanation

Extreme blue or red lines Sample with extremely in heatmap (when only low or high protein selecting "samples" in quantification the drop-down menu) or outlier dots in z-score view

NOTE: Extreme outliers can be discovered in different views (Plate QC -> Z-scores, Assay QC, Heatmap). In Figure 12, two types of outliers are illustrated.

Reason
Incorrect dilution of sample No sample added

Action Fail sample

Figure 12. Heatmap with display type Delta with NPX values from a single plate run which contains two outliers. Sample 25 has the lowest NPX values for all assays and Sample 71 has the highest NPX values for all assays.
42

For Sample 25, most NPX values are below LOD and one conclusion for this outlier is that there is no biological sample present in that well. For Sample 71 in this example, the NPX values are higher than for the rest of the samples. This can arise from poor dilution of a sample when running one of the high-abundant panels.
Figure 13. Ct-values that show results of laboratory mistakes. See Table 12 on page 44 for a description of errors performed on this run.
Figure 14. NPX values that show results of laboratory mistakes. See Table 12 on page 44 for a description of errors performed on this run.
43

Table 12. Explanation of columns in Figure 13 and Figure 14. Red numbers indicate laboratory mistakes.

Column 4 Column 7 Column 8 Column 9 Column 10 Column 11 Column 12 (correct)

Incubation mix (µL)

Sample volume (µL)

Extension mix (µL)

192

Detection mix (µL)

7.2

14.4

7.2

PCR product (µL)

2.8

5.6

Figure 15. Ct values that show primer contamination of Incubation Control 2.
Figure 16. Within plate effects due to incorrect or insufficient vortexing of the sample plate during sample dilution. 44

13.4.4 High NPX signals - common reasons

Issue

Explanation

Reason

Unexpectedly high NPX signals in diluted panel for the majority of samples

Low variation between samples Saturated assays (may be visualized in Z-scores tab)

Samples were not diluted

Action
Rerun from sample dilution step

14. Revision history

Version

Date

1.0

Description

15. Appendix A:

References

Adobe PDF Library 16.0.3 Adobe InDesign 17.0 (Windows)