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Table of Contents

Introduction
Welcome to Q-View Software
Terminology
Projects

Quick Start Guide

Q-View Installation
Software Installation
Imager Installation
Q-View Imager Pro Notes

Access Control
User Management
Password Policy
User Roles and Permissions
Electronic Signatures

Image Processing
Image Acquisition
Import Images
Manage Images
Align Plate Overlay
Auto-Set Plate Overlay
Auto Spot Finding
Assay Controls

Well Assignment
Assigning Wells in Q-View Software
Assigning Wells in a Spreadsheet
Well Assignment Templates

Data Analysis
Curve Fit Options
Regression Models
Weighting
Negative Well Subtraction
Viewing and Customizing the Data
Mask Outliers
Statistics
Assay Limits
Evaluating the Quality of the Curve Fit
Data Analysis Templates
Error Codes
Submit Report for Review
Approve/Reject Report
Export
Q-View Imager Qualification
Settings
Perform Qualification
Reports
Recertification
Build Ranges

Imager & Software IQ/OQ
Settings
Perform Qualification
Reports

Blot Tools
Selection Options
Illuminance Plot
Blot Report

Administrative
Software and Product Updates
Settings
Event Log
Project History
Uninstalling the Software

Support
Technical/Troubleshooting
Contact Quansys

Introduction
Welcome to Q-View Software

Figure 1. Q-View Software workflow.
Q-View™ Software is a tool for the quantitative analysis of multiplexed chemiluminescent planar-based arrays. Analysis is performed in three easy steps (Figure 1) which are covered in detail in this manual:
Terminology
Projects

Figure 2. Multiple projects opened.
Q-View Software stores the images and analyses of related plates together in a file, called a project, with the ".Q-View" extension. As you work, changes to these projects are saved automatically. Multiple projects can be open at the same time (Figure 2).

Before version 3.1, projects were stored as folders. Project folders can still be opened by the software but they will be upgraded to the project file format when opened. Project files are specific to Q-View Software and cannot be viewed or manipulated by 3rd party software.

Project details can be accessed by opening Project > Project Details. The project creator, creation date and a list of users that have opened the project along with associated timestamps are recorded.

Project notes can be recorded and viewed by opening Project > Project Notes. These notes allow users to record read-only messages related to the project.

Whenever a project is closed or a report is exported, the full report is recorded if it has changed in any way. Project history can be viewed by opening Project > Project History.

Quick Start Guide
  1. Installation
    1. Install Q-View Software on any number of computers.
    2. Set up your imager and any other equipment to be used for the experiment.
    3. Using a Q-View imager, ensure the imager has been focused, dark field images have been captured, and set-up is complete before proceeding. All of these procedures can be found in the Q-View Imager Pro and LS manuals.
  2. Process and image the Q-Plex kit
    1. Process the Q-Plex kit according to the instructions in the kit manual.
    2. Capture the image(s) using a Q-View imager or other compatible, third-party imager.
      With third-party imagers, only grayscale, TIFF and CR2 image files of at least 12 bits per pixel should be imported for scientific reporting purposes. Images with a white background must be inverted within Q-View Software by clicking Image Options > Invert Image .
  3. Analyze images in Q-View Software
    1. Enter the software product code (found on the kit product card) for each plate.
    2. Within Image Processing, set the plate overlay.
    3. Perform Well Assignment.
    4. Within Data Analysis, click Perform Analysis to generate charts and reports. Customize and export the results as desired.
Q-View Installation
Software Installation
  1. Ensure the computer meets the following minimum specification:
    • Windows® 7, 8 or 10
      Note: The Windows® version of the Q-View Software is required to operate Q-View imagers. The Mac® operating system is not compatible with Q-View imagers.
    • Mac® OS 10.11 or newer
    • 4 GB RAM
    • Screen resolution of 1280x800 or greater

    For optimal performance:
    • 8+ GB RAM
    • 2 or more processing cores
  2. If you do not have a Q-View Software installer, contact a Quansys representative by email at sales@quansysbio.com or call 1-888-782-6797.
  3. Launch Q-View Software. Click either Evaluate Q-View to start a 60 day trial or enter your assigned company name and license key then click Continue. Follow the prompts on the screen to set up user accounts and Q-View imagers. You can change these settings later from the Settings menu.
Imager Installation

Figure 3. USB and power sockets.
  1. Carefully unpack the Q-View imager and place it on a clean work area.
  2. Plug the power cord into the back of the imager (Figure 3) and into a surge protector.
  3. Plug the USB cord into the back of the imager (Figure 3) and into the computer. For the Q-View Imager Pro, ensure the power switch is in the on position.
  4. Clean the tray glass with a lint-free cloth and ethanol, rubbing alcohol, or window cleaner.
  5. Launch Q-View Software and open Settings > Manage Imagers to discover, name and focus the imager and to capture a dark field. Dark field capture takes approximately 15 minutes.
Q-View Imager Pro Notes
Access Control
User Management
When user accounts are enabled, users are required to log in to use the software. Administrators can manage users during software setup or by opening Settings > Administration > User Accounts (Figure 4).
Figure 4. User accounts.
Password Policy
Administrators can set their organization's password policy by opening Settings > Administration > Password Policy (Figure 5). The password policy defines the following password requirements:
Figure 5. Password policy.
User Roles and Permissions
Administrators can manage user roles by opening Settings > Administration > User Roles (Figure 6). User roles define permissions which allow or disallow users from accessing various software features.
Figure 6. User roles and associated permissions.
By default, users are assigned the Default role which allows all permissions. The default role permissions cannot be modified. Users can view their permissions by opening Settings > Permissions (Figure 7).
Permissions are granted by selecting the Allowed check box adjacent to the permission (Figure 6). Permissions are revoked by deselecting the check box. The permissions are described as follows:
Figure 7. User permissions.
Electronic Signatures

Figure 8. Electronic signature.
When documents are saved, electronic signatures (Figure 8) are required if user accounts are enabled. The name of the signee, a reason for the signature and a timestamp are appended to the document. Electronic signatures apply to the following:
Figure 9. Electronic signature settings.
Electronic signature settings (Figure 9) can be accessed by opening Settings > Administration > Electronic Signature Settings. You can create or delete signature reasons for specific categories. The order of the reasons can by modified by moving them up or down in the list.

When the Include 'other' option is selected, an 'other' option will be included in the reason drop-down list (Figure 8). A free-form text field will be enabled allowing the user to enter a custom reason. When the Require reviewer signature option is selected, exporting reports requires that they first be submitted for review by users with the Submit reports for review permission and subsequently approved by users with the Approve and reject reports permission.

Image Processing
Image Acquisition

Figure 10. Image acquisition options.
  1. Place the plate in the Q-View imager and close the lid/tray. Select the desired imager, exposure time(s) and image name(s) (Figure 10). If the desired imager is not listed, select Refresh from the drop-down to scan for imagers.

    The recommended exposure settings for Q-Plex kits are:
    • Q-View Imager Pro: 300 seconds
    • Q-View Imager LS: 270 seconds
  2. Capture the image(s) by clicking Capture Image(s). You can continue to Well Assignment while images are being captured.
  3. Add a plate tab for each physical plate to be imaged by clicking the + tab (Figure 11). Custom plate names can be set by double clicking the plate name.

Figure 11. Plates organized by tabs.
Note: The Enable Internal Illumination option is available for Q-View Imager Pro models when activated in Settings > Preferences. When enabled, the imager's internal focus lights are turned on during image acquisition. This may be useful for quality control but should not be enabled when capturing Q-Plex images.


Import Images
Before importing an image, ensure that it is grayscale and has a pixel depth of at least 12 bits. Q-View Software can open TIFF, CR2, BMP, PNG and JPEG files. Low bit depth and lossy images (BMP, PNG and JPEG) are insufficient for analysis and should be imported for display purposes only.

To import an image, click Image Processing > Import Image or drag and drop image files onto the list of images.
Manage Images

Figure 12. Image pop-up menu.
Images can be renamed, deleted or stacked by right-clicking an image name (Figure 12). Image details can be viewed by selecting properties. Click Export to save an image as a lossless TIFF or compressed JPEG. JPEG files are suitable for display purposes only and should not be used for analysis.
Align Plate Overlay
  1. Enter the Software Product Code from the product card into the Product Code field.
  2. Use Image Options to adjust image gamma, zoom and orientation as desired. Images with black spots on a white background need to be inverted by clicking Image Options > Invert Image.

  3. Figure 13. Overlay Options.
    If you are using the Auto-Set Plate Overlay feature, the initial overlay position will be set automatically. Otherwise, click Overlay Options > Set Plate Overlay, and click and drag the mouse cursor from the center of the top-left well to the center of the bottom-right well. Use the following Overlay Options (Figure 13), if needed, to manually adjust the overlay:
    • Set Plate Overlay: Set the plate overlay by dragging from the center of the top-left well to the center of the bottom-right well.
    • Adjust Plate Overlay: Pivot or expand/contract the entire overlay via clicking and dragging the corner wells highlighted in blue.
    • Adjust Spot and Adjust Well: Click and drag to move either an individual spot in the overlay, or all spots in a well. Use the keyboard arrow keys for more precise adjustments.
    • Auto-Adjust Spots: Automatically adjusts all spots to improve the alignment of the overlay.
    • Reset Spots: Reset all spots in the overlay to their default locations.
    • Hide Plate Overlay: View the image without the plate overlay.
Auto-Set Plate Overlay
When using a Q-View imager, the Auto-Set Plate Overlay feature, found in Settings > Preferences, can be enabled to cause the software to provide the initial plate overlay position.
Figure 14. Plate overlay.
This feature is activated after manually setting a plate overlay for the first time. Once activated, the Auto-Set Plate Overlay feature will automatically set the overlay onto all captured images as soon as the product code is entered. Be sure to review the accuracy of the overlay placement for each captured image.
Auto Spot Finding
When using a Q-View Imager Pro or Q-View Imager LS with Q-Plex plates, Auto Spot Finding will be active and will make it easier to accurately set the plate overlay either manually or via the Auto-Set Plate Overlay feature.
Assay Controls
For products with defined assay controls, the assay control spots that are in range are outlined in green. Spot(s) and associated well(s) that are out of range are outlined in red (Figure 14).

Well Assignment

Figure 15. Well assignment.

Assign well types, names, and dilution factors in either Q-View Software or a spreadsheet program such as Microsoft Excel. Use Well Assignment Templates to quickly assign repeat layouts.
Assigning Wells in Q-View Software
To assign well types and names, select the desired wells by clicking and dragging the mouse over the plate image (Figure 15). Hold the control key to select discontiguous groups of wells. Click Groups (Figure 16) and enter custom names if desired. Select one of the well types defined below to register the assignment.
Figure 16. Group tools.
Calibrators may be assigned dilution factors or grouped by predefined concentrations for each analyte. Predefined concentrations are created by Quansys and assigned by the user by selecting a well group, clicking the Calibrator button (Figure 16) and then choosing from a list of group names in a subsequent pop-up window.

Use Sequential Naming to automatically name groups of wells with sequentially increasing suffixes.


Figure 17. Dilution tools.
To assign dilution factors (Figure 17), select the desired wells and click the Dilutions button. Enter the dilution factor of the initial dilution in the Initial Dilution field. For example, enter the number "2" for a 1:2 (50%) dilution. If a dilution series was continued from that initial dilution, enter the series dilution factor. For example, to indicate a 1:10 dilution series from the 1:2 (e.g. 1:20, 1:200, 1:2000), enter "10" in the Dilution Factor field. Press any of the directional buttons, e.g. Top to Bottom, to register the assignment.
Assigning Wells in a Spreadsheet
To assign well types, names, and dilutions in spreadsheet software such as Microsoft Excel, click Templates > New Template, then name and save the newly created template file. Once saved, a blank well assignment template will open in the default spreadsheet software. Fill this out and save your changes. Apply the template as described below.
Well Assignment Templates
To save your current well assignment as a template, click Templates > Save Template. To apply a previously saved template to a plate, either click Templates > Open Template, or drag and drop the template file onto the plate diagram.

Data Analysis
Once you have completed Image Processing and Well Assignment, select Data Analysis. Click Perform Analysis to generate charts, concentrations, and statistics based on optimal regression settings provided by the product definition. Click the Curve Fit Options button to customize regression models, weighting, and negative well subtraction settings.
Curve Fit Options

Figure 18. Curve fit regression options.
The default 4PL or 5PL regression models are generally recommended for most Q-Plex data, as these are widely considered to provide the best overall fit for immunoassay curves. If your data is abnormal or you are trying to optimize the fit for a particular range, one of the other regression models, along with masking, may better fit your needs. You can choose from the following Curve Fit Options (Figure 18):

Regression Models In products with spot replicates, replicates within each well are averaged and treated as single data points. Calibrator curves for assays with spot replicates are fit to the replicate average using the specified regression model.

Calibrator curves for our High Dynamic Range Singleplex assays are automatically fit using the HDR algorithm irrespective of the chosen curve fit option.


Figure 20. Curve fit negative subtraction and weighting options.
Weighting

Weighting options are available after selecting Show Advanced Options. Weighting is used to reduce the impact of outliers and offset heteroscedasticity. In ELISA calibrator curves, the residuals (variability) tend to increase as the concentration of the calibrator (y) increases. To improve curve fit, various weighting methods can be applied. General weighting (y) can be applied to normalize the residuals across the curve based on the signal of each calibrator point. Weighting based on residuals (r), reduces the impact of outliers on the curve fit. The larger the number associated with either weighting method, the more variation there will be between the impact of the calibrator points on the final curve fit.

Curve fit can be evaluated by looking at the percent backfit value in the report. When samples fall at the low or high end of the curve, goodness of fit should be prioritized at that end of the curve. Selecting Favor Low or Favor High evaluates the percent backfit across the curve and will automatically select the weighting method that results in the best fit for either the low or high end of the curve.

Negative Well Subtraction

Negative well subtraction (Figure 20) automatically deducts the average signal (pixel intensity) of the negative wells from the signal of all other wells for each analyte. Curve fit and calculated concentrations are not affected by negative well subtraction. The default state, on or off, can be changed in Settings > Preferences.
Viewing and Customizing the Data
Concentrations, pixel intensities, and statistics are displayed based on the selected Data Analysis tab (Figure 21), spot masking, and settings in the Statistics and Assay Limits menus.

Results are organized into three tabs for each plate:
Figure 21. Data Analysis tabs.
Other values that may appear in the Report and Data tabs are:
Mask Outliers

Figure 22. Masking spots.
Select and right-click (Figure 22) data points within the Chart, Report or Data tab to mask (exclude from all calculations) or unmask spots and wells.

Q-View Software can automatically identify and mask outliers in calibrator curve data. To enable this feature, open Settings > Preferences and select mask calibrator outliers by default. When you unmasked an auto-masked outlier, Q-View Software will not auto-mask the point again when fitting curves until you right click a chart or report and select Mask Outliers (Figure 22) from the drop-down menu.
Statistics
Click Statistics (Figure 23) to select which calculations are shown.


Figure 23. Statistics and Assay Limits.
If multiple dilutions of a sample were present, select Auto-Select Dilution to view only the data for the dilution that fell on the most linear part of the calibrator curve, instead of data for every dilution of the sample.
Assay Limits
Click Assay Limits (Figure 23) to modify how extrapolated data is displayed:
Evaluating the Quality of the Curve Fit
Backfitting of the calibrator is a practical method for evaluating the quality of the curve fit. This is done by comparing the calculated concentration of each calibrator to its known (theoretical) concentration using the equation: calculated concentration / theoretical concentration x 100%. Q-View Software displays this result in the report as percent backfit. The preferred result for each calibrator point is a backfit value of 80%-120%, although 70%-130% may be acceptable.
Data Analysis Templates

Figure 24. Data analysis templates.
Custom curve fit settings, statistics and assay limits can be saved in the Data Analysis Templates section (Figure 24). To save your current settings as a template, click Save Template. To apply a saved template, click Open Template or drag and drop a template file onto the data analysis section.
Error Codes
The error code column in the report alerts you to potential problems with your assay. Error codes are defined at the bottom of the report and include: Assay Controls (AC), Process Controls (PC), Spot Overlay Out of Bounds (SB) and Masked Outliers (MO).
Submit Report For Review
When User Accounts are enabled and the Require reviewer signature option is selected, reports are required to be submitted for review (Figure 25) and subsequently approved before they can be exported. Users are required to have the
Figure 25. Submit for review.
Submit reports for review permission to perform this action. If a report changes after it has been submitted for review, resubmission for review is required. This does not apply if the user who modifies the report has the Approve and reject reports permission.
Approve/Reject Report

Figure 26. Review report.
Users with the Approve and reject reports permission can approve or reject reports (Figure 26) submitted for review. Rejected reports must be resubmitted for review and approved before they can be exported. Approved reports can be exported by users who have the Export data analysis permission.

Figure 27. Export.
Export
Click Export (Figure 27) to save charts as images, or the currently displayed values in the Report or Data tabs as a spreadsheet (CSV).

Q-View Imager Qualification
The Q-View Qualification Plate produces a constant amount of light at intensities across the dynamic range of the imager. Qualification captures images of the reference plate and evaluates performance based on defined specifications. Performing qualification on a regular basis ensures that the imager consistently meets performance specifications.
Settings

Figure 28. Q-View Imager Qualification menu.
Q-View Software imager qualification settings (Figure 29) can be accessed from the Qualification > Q-View Imager Qualification > Settings menu (Figure 28). The settings are accessible to only administrators.
Perform Qualification

Figure 30. Image capture is restricted.
Power on the Q-View Qualification Plate and place it in the imager face down in the lower right corner of plate deck. Select Qualification > Q-View Imager Qualification > Perform Qualification from the menu (Figure 28) or click Q-View Imager Qualification has expired (Figure 30) from the Image Acquisition screen. Confirm that the serial number on the qualification plate matches the serial number in the window. The process takes about 5 minutes to complete.
Reports
To view and export qualification reports, select Qualification > Q-View Imager Qualification > Reports (Figure 28) from the menu.
Recertification
Qualification plate certification expires annually. The expiration date is shown on the back of the plate. Once certification has expired, please contact Quansys Biosciences to arrange to have your plate recertified. Once your plate has been recertified and returned to you, it is necessary to rebuild the qualification ranges.
Build Ranges
To build qualification ranges, open Qualification > Q-View Imager Qualification > Settings > Build Ranges and follow the instructions provided by the software.

Imager & Software IQ/OQ
IQ/OQ testing ensures that the installation and operational functions of the imager and software are performing correctly. These tests can be used upon initial evaluation and subsequent evaluations to ensure consistent operation of the Q-View imager and software. Following the IQ/OQ testing, a report is generated that may be used for regulatory compliance and equipment control.

Figure 31. Imager & Software IQ/OQ menu.
Settings
Imager & Software IQ/OQ settings (Figure 32) can be accessed from the Qualification > Imager & Software IQ/OQ > Settings menu (Figure 31). The settings are accessible to only administrators.
Perform Qualification
In the Q-View Software, select Qualification > Imager & Software IQ/OQ > Perform Qualification from the menu (Figure 31) or click Imager & Software IQ/OQ has expired (Figure 33) from the Image Acquisition screen. The process takes about 15 minutes to complete.
Figure 33. Image capture is restricted.
Reports
To view and export qualification reports, select Qualification > Imager & Software IQ/OQ > Reports (Figure 31) from the menu.

Blot Tools
Click Support > Legacy Blot Tools to make custom selections for western blots, northern blots, southern blots, dot blots, and other chemiluminescent images. Click Close to return to the previous Image Processing screen.
Selection Options
Click Selection Options and choose one of the following tools to make a custom selection (Figure 34).


Figure 34. Blot tools.
Auto Select: Automatically select a group of similar, contiguous pixels by clicking on the center of the grouping. The number of shades included in the selection correlates to the sensitivity value (0-100) entered in the Sensitivity field.


Figure 35. Auto select tool. On the top, only the outer edge is selected, whereas on the bottom, the entire area is selected. The light red fill indicates the selection boundaries.
The selection is indicated by a bright red border and filled with translucent red while unselected areas are indicated by a translucent gray fill. Make sure the intended pixel grouping is selected, rather than just a ring around the grouping (Figure 35).

Rectangle and Ellipse: Create a rectangular or circular selection by clicking and dragging the location where the selection is to be placed.

Polygon: Draw a custom, straight-edged selection by clicking on the image where each vertex is desired. Right-click to close the polygon.

Selections can be moved by dragging with the mouse, cleared with Clear Selection, and exported as a JPEG image by clicking Export As Image.

Manipulate the selection boundaries by clicking and dragging, or use the following keyboard commands.

Tool Arrow keys move selection Alt + arrow keys to lengthen/shorten shape +/- keys to resize
Auto Select
Rectangle
Ellipse
Polygon
Note: Hold shift to amplify actions.
Illuminance Plot

Figure 36. 3D illuminance plot.
To create a plot of the illuminance of a selected area (Figure 36), click View 3D Plot. The 3D plot will appear in a new window. The plot can be viewed at any angle by clicking and dragging. Hold control while dragging to move the plot. Roll the mouse wheel to zoom in and out. Various plotting options are available on the right side of the window.
Blot Report
The Blot Report is an exportable table that includes custom names, number of pixels selected, mean pixel intensities, and standard deviations. To add a selection to the table, click Add to Report. You can add as many entries as you would like. The information for the current selection is shown at the bottom of the drop-down. The table can be viewed and edited at any time by clicking View Report. To view the pixel coordinates and intensities for a selection, click the blue # Pixels label.

Administrative
Software and Product Updates

Figure 37. Update options.
Both software and product updates can be obtained automatically if set to do so in Settings > Administration > Q-View Updates (Figure 37). If updates are not occurring, your network settings may need to be modified in Settings > Administration > Network Settings.

If your computer does not have Internet access, contact Quansys at sales@quansysbio.com or 1-888-782-6797 to arrange for regular software updates to be sent to you.

To apply product updates manually, go to Settings > Administration > Q-View Updates and select Update product definitions manually, then click the Update product definitions from file button. A file selection window will appear. Navigate to the product definitions file you received from Quansys and select Open.
Settings

Figure 38. Administrative options.
Use the options under the Settings menu as follows (Figure 38):
Event Log

Figure 39. Event log.
A variety of events and setting modifications are recorded in the event log. The event log (Figure 39) can be accessed under Settings > Administration > Event Log. Events can be filtered by type, user and date. The log can be exported by clicking the Export button.
Project History

Figure 40. Project history.
Whenever a project is closed or a report is exported, the full report is recorded if any changes exist. Project history (Figure 40) can be accessed by opening Project > Project History. By selecting two entries in the history list, you can compare reports to identify differences. Differences are highlighted in yellow. Reports can exported by clicking the associated Export button.
Uninstalling Q-View Software
Q-View Software can be uninstalled through your computer’s settings or by opening the Q-View Software installer and clicking Remove.

Support

Figure 41. Help button.
This manual is available in the software by clicking the help button (Figure 41), under Support > Q-View Software Manual, and online at www.quansysbio.com/manuals.
Technical/Troubleshooting
Tips for many aspects of using multiplex ELISA's are available online at www.quansysbio.com/tech-tips.

Technical support data such as projects and imager databases can be sent to Quansys Biosciences directly from Q-View Software by opening Support > Technical Support Upload and selecting either Upload Project or Upload Imager Database. If your computer is not connected to the Internet, the upload options will prepare the data for manual upload which is accomplished by moving the produced file(s) to a computer with Internet access and visiting support.quansysbio.com.
Contact Quansys
To contact Quansys, please:
Call us at 1-888-QUANSYS (888-782-6797)
Chat with us online at www.quansysbio.com
Email us at sales@quansysbio.com for software or other product requests
Email us at support@quansysbio.com for technical and troubleshooting issues
Email us at info@quansysbio.com for other general inquiries