LEGENDplex Data Analysis Software

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LEGENDplex™ Data

Analysis Software

Version 7.0

User Guide

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Introduction

LEGENDplex™ Data Analysis Software is a quantitative analysis package for FCS data

generated by a flow cytometer. It is designed to provide a user-friendly interface, accurate analytical results, and automated data report.

LEGENDplex™ offers:

 Easy-to-use ribbon style user interface

 Simple wizard for repetitive tasks required to analyze flow data

 Fully automated analysis including data reduction and concentration calculation  Robust curve fitting and accurate detection limit determination

 Completely integrated visualization between original data, curve mapping and results

 Standardized reports with graphic presentation  Support both quantitative and qualitative analysis

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Lesson 1 - The Workspace

LEGENDplex™ is similar to Microsoft Office products, which places a greater emphasis on visual orientation to software features to facilitate ease of use. The hierarchy is that Ribbons contain Tabs, Tabs contain Groups, and Groups contain Tasks.

Click to hide/display the tabs details.

Figure 1. LEGENDplex™ Ribbon

Ribbons

Ribbons contain both tabs and groups, along with access buttons for different functions.

Tabs

Tabs are a set of groups, organized by similar function.

For example, the Analysis tab contains the Wizard, Preference, Edit, Analysis,

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Groups

Groups are a set of similar features or program tasks similar to menus, but with images, and tool tips.

For instance, the View group contains 3D, Bar Chart, Standard Curve and Sample Detail. These are all visual presentations of results but in different forms.

Lesson 2 – Quantitative Wizard

1. Click to start a new quick wizard session.

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4. Click Next and adjust the parameters on the Settings panel according to the assay. In the example shown above, we set Highest Conc. to 4000, Dilution Factor to 4, and Decimal Places to 0.

The AABBCC mode will replicate samples in consecutive manner when the number of replicates is defined (e.g. sample 1, sample 1; sample 2, sample 2; …..). The ABCABC mode will replicate samples in repeating groups with defined group size (e.g. sample 1, 2, 3, 4…; sample 1, 2, 3, 4…).

It is recommended that the Auto Gate be set at OFF position.

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5. Click Next. Sort the loaded files to the order you need by clicking the column titles File Name, or Data modified in the file list, the program will sort the files in ascending/descending order.

In your experiment, if you follow the LEGENDplex™ kit Plate Map or Rack Map and acquire data for standards and samples in the same sequential order as that in the Plate Map, you can do file sorting by clicking on Date modified to put files in correct order, select the consecutive files and then click on to apply the standard curve parameters according to the default Settings.

Note: You should click to apply the curve options whenever a parameter of Standard Curve settings has been altered.

If the FCS files for the standard curve are inconsecutivein the list, you can press and hold down the Ctrl key and select the files in the correct order of the standard curve, then right click Apply Curve Options.

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replications and dilution factors, you may highlight selected samples, then right click your mouse and go to Edit Samples functions.

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B4…).

You will need to select the X-axis and Y-axis labels (channels) of the bead dot plots. The channels vary depending of the flow cytometer used to collect the data. The default settings are for the BD FACSCaliburTM instrument.

Then click on the top of the Bead Gating window, LEGENDplex™ will find bead areas automatically.

For LEGENDplex™ beads-based assays, the auto gating works well if you follow the kit protocols and the flow cytometer used for sample reading is properly set up and compensated (if needed). If for some reason autogating failed to properly separate the bead populations, please use following options:

a. Decrease Gate Setting (e.g. from 100 to 50), or

b. Use manual gating: You may need to click on and then click on the bead population to remove the gate set by auto gating. Click and then draw a gate around the bead size population(s). The new gating will automatically apply. If you find that the Band Panel (lower panel) is not acceptable, you may also use

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to erase the rectangular gate for a particular population and re-draw a new gate using .

After manual gating if you click on , it will remove all manual gating.

c. Use a different sample to do gating. This is usually the most practical method.

You can click to save current gate settings to a gating protocol.

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Standard curves and data calculation options can be edited using the two buttons labeled as Curve and Options.

Variations in standard concentrations for different analytes can be modified in the Curve and Options. Curve fitting method can also be changed in Options. If you forget to define files for standard curves in Step 5, the Curve and Option button may not work. You will need to click on the Previous button to go back to the previous page to define the files used for standard curves.

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The result will be displayed as shown below.

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10. Click Report on the Ribbon menu and select a report type to generate a report. The report can be printed directly or exported to Excel, Word or PDF files.

Lesson 3 – Qualitative Wizard

1. Click to start a new quick wizard session.

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3. Select the .fcs files or .lmd files (a special form of .fcs files) and drag them to the FCS Files list. You can also drag the file folder to the FCS Files list;

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5. Click on the bottom left of the window to do gating procedure. 6. Click Next to go to the next step.

7. Do the followings if necessary.

 Edit Sample ID

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9. The result will be displayed as below.

The results include Normalized MFI, Median CV, Median, and Count. Click tabs to switch between the views.

Lesson 4 – Selected Functions

More detailed functions of the software are described below.

Load Shared Files

LEGENDplex™ allows the users to load files on a network. However, a network drive may need to be mapped first. To map a network drive, follow the instructions below. 1. Open Computer by pressing Win+R or double click the Computer icon on your

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2. Select Map network drive marked in the above picture to open Map Network Drive dialog box.

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4. In the Folder box, type the path of the folder or computer, or click Browse to find the folder or computer.

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Set Standard Curve Manually

If the FCS files for the standard curve are in the middle of the file list, you may drag the small round buttons labeled as C0, C1, C2….. on top of the file list individually to appropriate files corresponding to the standard points (such as C0 for the 0 Standard, C1 for the lowest standard concentration, and C2 for the second lowest concentration of the standard curve, etc.).

You can also select C from the drop down list of Type column in the FCS Files list and manually type a concentration in the Concentration column.

The designates a sample and to ( please change to C0 to C7) indicates calibrators (i.e. Standards). Move the mouse over the icons, a tooltip will display the theoretical standard concentration according to Quantitative Settings.

Alternatively, the files corresponding to Standards can be highlighted and right clicked to change file types to C. The standard concentrations can be typed in the Concentration column.

Data Sorting

In the FCS Files list, the column title such as File Name, and Data modified can be used to sort the files in ascending/descending order. Common use of the sorting function is to arrange the order of files for standard curves to match with experimental layout so that data can be calculated correctly.

Replicates

Sample replicates can be defined in two ways:

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2. Highlight samples, right click in the FCS Files list and select Edit Name to open the Define Names dialog box. Define the Replicate Mode and Replicate

Number.

Define Sample Dilution

Select sample files, right click in the FCS Files list and select Edit Name to open the Define Names dialog box. Input the Dilution value. Or you can input the dilution value directly in the Dilution Fold column of the FCS Files list for each sample.

Edit Sample ID

Select a file or multiple files in the FCS Files list, then right click in the list and select Edit Names to open the Define Names dialog box.

Prefix --- Enter a Prefix of sample well name series (The default is Sample). Start Number --- Enter a starting number of sample name series.

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Replicate number --- Enter number of replicates for the selected samples.

Dilution Factor --- Enter a Dilution Factor if the sample is diluted.The software will report sample concentrations adjusted for dilution factors in the final report.

Flag and Unflag

Flag data:

 Select one or a group of data, right click the data and select Flag from the popup menu, or

 Click on the Analysis tab.

Unflag Data

 Select one or a group of flagged data, right click the data and select UnFlag from the popup menu, or

 Click on the Analysis tab to cancel the flag.

Edit Standard Curve

Standard curve can be further edited prior to analysis. Step1: Open Edit Standard Curve dialog box.

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Step2:

 Verify or redefine the standard curve in the left panel (Standard Curve).

 Select the check boxes in front of the IDs. If the check box is blue, it means that it is the replicate of the well above.

Step3:

 Enter a value for the highest concentration, dilution factor for the standard curve and the number of decimal digits for the calibrated sample concentrations.

 Check Background (C0) check box to indicate that the standard curve files include background wells. Uncheck it to indicate the standard curve files do not include background files.

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shape). Increasing is used most often with typical sandwich assays involving S shaped curves. Decreasing is often used for competition assay curves.

 If all analytes have same starting concentrations, select Apply to All Analytes check box (default). Click button to continue.

 If not all analytes have the same starting concentrations, uncheck the box for Apply to All Analytes, select the analytes which share the same concentrations, enter the concentration in the Highest Concentration box, and then click

for these selected analytes. Repeat the same operations for other targets which have different highest concentrations. When all analytes are defined, click OK button.

Set Control Type

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Reanalyze

Lesson 5 - Gating

Gating is the process of separating bead populations. Bead size populations can be further gated to generate subpopulations. LEGENDplex™ Data Analysis Software offers

automatic tools and advanced manual gating tools to facilitate this process.

First, open the Bead Gating window. Select any sample and click on the left bottom or the same icon on the Analysis ribbon tab at Wizard step 6.

Gating Toolbar provides a series of tool to finish the gating process. Gate Setting is where to define the gating parameters.

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Gating Protocol

Gating protocol is a .gating.xml file which contains gate setting and gating results. You can load a saved gating protocol file by clicking to browse.

Also you can click to save current gating setting and apply gating results to a gating protocol.

Auto Gating

Adjust the value of # of Bead Size, #Bead of Size 1, Size 2 and change the x and y axis parameters of the Size Panel and the Band Panel to match the experiment.

To define the size, choose SSC-H in the y axis and FSC-H in the x axis of the Size (upper) panel. To define the subpopulations of beads, choose FL3 or FL4 in the y axis and FL2 in the x axis of the Band (lower) panel. The selection may vary with data

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Then click on the top of the Bead Gating window, LEGENDplex™ will find bead clusters automatically.

Manual Gating Tools

If there is severe background noise which may affect the gating process or the gating results don’t match your expectation, LEGENDplex™ provides a set of tools to perform

gating manually.

Add a polygon cluster in Size Panel or a rectangle in Band Panel.

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Lesson 6 – View

3D Result View

To display results in 3D view, highlight selected Concentration or Median data and click on the View ribbon tab, the 3D View Window will be displayed as below. The 3D view can be rotated or zoomed with your mouse. The 3D image can be saved or copied to other applications by right clicking your mouse and select the Save or Copy function.

Bar Chart View

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When the mouse moves over each bar, a tooltip will display the specific value. If too many data points are highlighted, the bar chart may not have enough room to display properly. You can enlarge the display window to properly display the chart.

StandardCurveView

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Sample Detail View

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Select the analyte (e.g. d) and sample(s) (e.g. F026) on the top of the window to display the corresponding sample detail view. Sample will be mapped in the standard curve. All sample concentrations can be mapped to a standard curve.

Clustering view or Heat Map

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Lesson 7 – Report

LEGENDplex™ offers 2 report types: Detailed Report and Summary Report.

Select a report type from the drop down list of on the Main Toolbar. The reports can be exported in Excel, PDF, or Word format.

Detail Report

The Detailed Report includes sample concentration details, standard curve details, file information, standard curve montage and signature.

Summary Report

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Contacting VigeneTech

For technical support and product information of LEGENDplexTM Data Analysis Software, please contact VigeneTech customer support and technical support. Web site www.vigenetech.com

E-mail [email protected]

Telephone 1-978-371-5959