Getting Started with Kaluza: Statistics

Here you will learn what options you have to display statistical results in Kaluza Analysis Software *, how you can perform more advanced calculations and how to export statistical results from Kaluza.

In flow cytometry, experiments often include the determination of the frequency of cellular subsets or of other statistical results such as mean fluorescence intensities. Kaluza offers several ways to display statistical results (Figure 1).

Overview of statistic results display in Kaluza

Figure 1. Overview of statistic results display in Kaluza. A) Gate Statistics displayed in gate label B) Statistics set up using Statistics Menu below the plot C) Gate Statistics Table offering basic statistic results and optional information on gate color and gating hierarchy D) Information Table customized to display statistic results. Plots are for illustration purposes only.

Outline

For detailed instructions, refer to Kaluza Analysis Flow Cytometry Software IFU C10986. This document does not replace Instructions for Use. Data for this Technical Note was generated using PBMC samples stained with dry reagents tube, acquired on a CytoFLEX LX cytometer* (PN: C40324) and analyzed using Kaluza Analysis Software*. 
Plots are for illustration purposes only.

Gate Statistics displayed in gate label

To get quick insights in basic statistical results it can be helpful to display these results directly at the gate they relate to (figure 2). To achieve this in Kaluza:
  • Create plots and gates to identify the population of interest. For detailed instructions on how to create plots and gates please refer to Kaluza Analysis Flow Cytometry Software IFU C10986.
  • Right click on a gate and access the Radial Menu > Display to change how the gate is displayed. The Statistic drop-down is where you can choose to display a statistic directly on the plot. Statistic options include All, Number, % Total, and % Gated. You can also choose to include a statistic label.
  • Select the Statistic drop-down list and choose the preferred statistic(s).
  • If you wish to show a label, select the check box located next to Show Label.
  • Select X to complete the process or click outside the dialog box.
Kaluza Statistic options in Display menu
Figure 2. Statistic options in Display menu.

Statistics below the plot set up using Statistics Menu 

Displaying statistical results below a plot will allow you to choose from a wider selection of statistics (figure 3).

  • To choose statistics to display at the bottom of the plot, access Radial Menu > (Statistics), which provides access for selecting statistics to display on the plot.
  • Make your selection(s) using the buttons and/or check boxes.
    Options include:
    • Population Statistics that allow you to choose statistics related to all events within a plot, including Number, % Total, and % Gated.
    • X Statistics/Y Statistics that allow you to choose statistics related to the X- and Y-axes, including Median, Arithmetic Mean, Mode, Standard Deviation, CV, Half Peak CV, Minimum, Maximum, and Geometric Mean.
Kaluza Statistics menu with Statistics Options dialog window

Figure 3. Statistics menu with Statistics Options dialog window.

TIP FOR SUCCESS:

Showing the Absolute Count statistic requires input before the calculation can be made. If the Cells/μL (Absolute Count) statistic is selected prior to configuring the Absolute Count Settings in the Protocol Options pane, the value appears as ### on the plot.

Statistics Visible displays chosen statistics on a plot. When deselected, any statistics you previously chose will be retained, but will not display on the plot. By default, Statistics Visible is selected.

Show the "All" Gate allows you to choose if the All gate statistics are visible. The “All” gate includes all events on the plot. Individual gate check boxes allow you to choose what gates have visible statistics.

Statistics Options allows you to make systemic changes to the appearance of statistics, including using thousands separators in whole numbers and/or fractions and choosing between 0 and 4 decimal places in fractional numbers and/ or percent.

Gate Statistics Table 

The Gate Statistics table provides a listing of selected statistics for the defined gates (figure 4). By default, gates appear in hierarchical order. 

Kaluza Gate Statistics Table

Figure 4. Gate Statistics Table

  • From the Plots & Tables ribbon tab, select the Gate Statistics icon, and drag it to the preferred location on your sheet.
  • To choose the gates and specify the order in which they appear:
    • Right click within the empty table and access Radial Menu > Data
    • If working in a Composite Protocol, choose the Data Set for the table by selecting the Input Data Set drop-down, and then choosing the desired Data Set.
    • To select the gates that appear on the table, choose from the following options:
      • Add All Gates: Adds all gates associated with the Data Set.
      • Remove All Gates: Removes all gates previously set up for the table.
      • Add All Colored Gates: Click this button to only add the gates that have been assigned a color.
      • Select Gates: Allows you to manually specify the gates to show on the Gate Statistics table. To make specific gate selections:
        1. Select the Select Gates hyperlink. A pop-up menu appears with gate categories.
        2. Hover your mouse over the gate type you wish to add to the Gate Statistics table and, from the additional pop-up window, select the gate(s) you wish to add to the table. Continue this process for each type of gate you wish to add.

The following video shows examples of how statistical tables can be managed in the Kaluza analysis software.

TIPS FOR SUCCESS:

  • You can use the Gate Statistics Table to show the Boolean gate logic for gates by selecting Show Logic.
  • Gates can also be added to the table, as follows:
    • Select gates from within the Color Precedence pane, and then drag and drop them in the table.
    • Press the (Alt) key, while selecting a gate from a plot, and then drag and drop it into the table.
  • To select the order in which the gates appear on the table, choose from the following options:
    • Color Precedence: Gates appear in the same order that they appear in the Color Precedence pane.
    • Alphabetical: Gates are listed in the order that they would appear in the alphabet.
    • Hierarchical: Gates are listed showing parent/child relationships.
  • To add statistics to the Gate Statistics table position your cursor over the table, right click, and access Radial Menu > Statistics
    • To enable gate statistics on the table, select the Statistics Visible check box.
    • To choose the Population Statistics to show on the table, choose check box corresponding to the desired option(s).
    • Parameter Statistics let you choose to include values for one statistic that will display for individual parameters for each gate.
      • In the Statistic drop-down, choose the desired statistic to show for the parameters.
      • Choose the Select Parameter link, and then select the desired parameter(s) from the pop-up menu(s)/sub-menu(s).

Information Table customized to display statistic results

Information Tables (figure 5) and Information Sheet (figure 6) allow you to include Data Set information, such as FCS keywords, statistics, and/or formulas in your Analysis. Information Tables work well for presenting data that contains a Name and Value only (such as keywords or statistics), as this table is limited to two columns. Similar to a spreadsheet application Information Sheets are easy to use and allow for a high degree of customization. You may add as many columns as needed, and you have control over the width of the column, which works well for entering formulas and text.

Kaluza Report Sheet with Information Table

Figure 5. Report Sheet with Information Table

Information Table

  • From the Plots & Tables ribbon tab, select the drop-down arrow on the (Information) icon, and choose Information. The table is added to the bottom of the sheet.
  • To add Keywords to the table
    • Click the <Add Keywords> hyperlink on the lower left side of the table. The Add Keywords configuration window appears.
    • If working in a Composite, select the Input Data Set drop-down, and then choose the Data Set to draw the keywords from, or select All Data Sets to include a copy of the keyword from each Data Set.
    • Select the option for the type of data to appear with the keyword value (keyword, description, or both). 
NOTE: Other than parameter Names and Descriptions (which always appear as defined in the Parameters pane) keywords cannot be changed; they appear exactly as derived from the source file.
  • To add Statistics:
    • Click the <Add Statistic> hyperlink to access the Add Statistic configuration window
    • Select the desired options from the Add Statistic window. Options include:
      • Show Data Set: Choose this check box if you want to include the name of the Data Set with the statistic.
      • Show Gate: Choose this option if you want to include the gate name with the statistic.
      • Input Data Set: If working in a Composite, select the Input Data Set drop-down, and then choose the Data Set from which to show statistics, or select All Data Sets to include a copy the statistic from each Data Set.
      • Input Gate: If applicable, choose the input gate for the statistic.
      • Statistic: From the drop-down, choose the statistic to show on the Information table entry. Once the statistic is selected, the Parameter section expands, allowing you to choose the parameter.
    • Under Parameter, select the desired option(s) for information shown with the statistic in the table (choose any, or all, of the following):
      • Show Name: Shows the parameter Name.
      • Show Description: Shows the parameter Description.
      • Show Scale: Includes the scale type with the parameter.
  • To add a Formula:
    • Right click on the table and access Radial Menu > Data.
    • Select the (Add a row to the information table) button.
      Every cell in the table can contain a unique formula, which can be used to show keywords, statistics, concatenated text, and perform calculations such as, addition, subtraction, multiplication, and division.
    • In the Name field, enter a name for the formula.
    • In Value, enter the formula, which will display as the calculated value on the table.
The following video shows examples for the creation of information tables and information sheets.

Information Sheet

The Information Sheet is a highly customizable table that has a similar appearance to a spreadsheet. This table allows you to enter formulas and text directly into the cells, or you also have the option of copying content directly from a spreadsheet application after the column Captions are configured to match the column titles in the spreadsheet.

Kaluza Information Sheet example

Figure 6. Kaluza Information Sheet example
 
For details on how to create an Information Sheet and to configure the content, please refer to Kaluza Analysis Flow Cytometry Software IFU C10986.

Exporting Statistics

Statistics from the following locations can be exported into a *.csv spreadsheet (figure 7):

  • Plots, through accessing Radial Menu > (Statistics). 
  • A Comparison plot.
  • An Information table or Information Sheet.

To export statistics from the selected Protocol(s) (statistics must be set up prior to completing this procedure):

  • On the Analysis List, select the row(s) from which you wish to export statistics.
  • Select > Export > Export selected statistics to export all plot sheets and report sheets in the selected Analysis List row(s).

OR

  • Select > Export > Export statistics in current sheet to export statistics from the current sheet only. The Export Statistics window appears.
    If the export is only for one item, you have the option to enter a name. If this pertains to the current export, enter a file name in the File name field.
  • Browse the folder where you wish to save the *.csv file(s) and click OK.
Kaluza Analysis List menu options including Export selected statistics

Figure 7. Kaluza Analysis List menu options including Export selected statistics

TIPS FOR SUCCESS:

Kaluza Analysis can produce up to three .csv files per Analysis, the number depending on which applicable plots/tables are included. When multiple .csv files result from exporting statistics, the files are automatically saved with the following format:

  • Plot statistics: datasetname_protocolname_statistic.csv
  • Comparison plot: datasetname_protocolname_comparison.csv
  • Information table: datasetname_protocolname_information.info

 

* For Research Use Only. Not for use in diagnostic procedures.