In FlowJo's Layout Editor, you can create a layout based on graphs and statistics from one of your samples. With a single click, you can ask it to produce the same layout from all of the rest of your samples.  Or any subset that makes sense for your experiment.

There are a couple of terms that we use in this explanation that are important to get clear from the start.  They are attribute and value. The keywords in an FCS file are organized in attribute-value pairs.  The attribute is a the keyword that is found in most files.  The second is the value of that keyword in any given file.  For example, most files will contain, in their header, a $Date attribute, and a value containing the date on which that sample was collected.  When we talk about iteration attribute we mean the attribute that is changed for each frame of the stack that is generated, for instance the date of the collection.

In advanced reporting, there is an additional feature known as the discriminator. This field is the mechanism for finding the correct sample, when more than one sample are collected with the same iteration value. This is described in more detail at the bottom of this page.

Generally, you would select a keyword that groups sets of tubes into distinct units--for example, choosing a keyword that connotes patient ID, tissue type, or experimental condition.  Then you can place multiple graphs from different tubes (from different FCS files!) into the same layout and still generate a batch output.

For example, if you set the iteration attribute to be the keyword "$Cells", where you have a Patient ID entered into the $Cells keyword, then FlowJo will examine all of the tubes in the current workspace, and generate a list of the unique values of $Cells.  These are then displayed in the "Iteration" popup menu.  By selecting any of these values from this menu, FlowJo will change the current layout view to show graphs & statistics drawn only from tubes with that value of the Iterator.  (By setting this popup menu to "Off", you are telling FlowJo to use all of the original graphs that dragged and dropped into the layout).  This is the way in which you can build template reports which select graphs and statistics from different tubes and put them all on the same page, but retain the ability to batch the outputs.

The most common case remains that where the iteration attribute is the sample (by which we generally mean the tube run through the cytometer, as opposed to the sample taken from the subject).

Iterating in the Layout Editor

The first thing to be aware of is what is the currently active group in the Workspace window.  This will be the selected item in the top half of the window.  This is important to know because the current group will determine the set of samples, from which the layout will be constructed.

Next you should look at the popup menu in the layout.  In most cases, you'll probably be iterating by sample, and the popup will appear like this:       

Changing the Iteration Value

There are several ways to change the iteration value.  You can use the popup menu in the layout editor window to show the layout for any value in the current group.  Alternately you can use the yellow up and down arrows to move to the previous or next values.  From the keyboard, you can use the home, page up, page down and end keys to move to the first, previous, next and last iteration values respectively.

Setting the Iteration Attribute

If you want to edit the iteration attribute, then select the command from the Layout menu called Iteration Options… This will bring up the following dialog box:

Group tubes by Numerical Order is the easiest method of iterating. If you want to overlay two tubes and batch process the layout so that tube 2 is overlayed on tube 1 and then tube 4 overlays tube 3 etc. choose Iterate over every 2 samples (tubes).

For more complicated iteration, choose Group tubes by Keyword value.

The popup menu in this dialog lists all the keywords found in all of the samples of the current group.  Select the keyword that you want common to all images in a frame of your layout, but to vary through each of the frames in the stack.

Multiple sample Iteration

The Iteration Options also provide the ability to specify one or more discriminator keywords. These fields provide the value that must stay constant, while the iteration attribute changes through each of its successive values. For example, if you have a ten mouse study, and for each mouse you collect three different tissue types, then there would be thirty tubes in the experiment. You want to produce a ten page report, showing the graphs and statistics for each mouse, one per page. You've acquired the data such that the mouse identifier is stored in the FCS file's $SMNO (sample number) field, and the tissue type information is stored in the $CELLS field. Set the field $SMNO to be the iteration attribute, and the keyword $CELLS to be the discriminator.

More than one discriminator can be defined if desired. By selecting additional keywords in the popup menu, the string of discriminators will be extended. To start over, choose the second item in the menu, marked Select None. This empties the list.