How to

How to use:

1. Prepare the data to convert in a regular ascii file. In Excel, choose "Text (tab-separated)". You can also prepare the data simply in a text editor. The data should be arranged such that each event is a separate line, where each parameter of the event is separated by a delimiter (such as a tabulator). An "event" means the single unit of study, for example each patient or each cell of the analysis. "Parameters" are the separate data that were collected for each subject, for example age, weight, height, etc. The data can be numerical, like height etc. Here, it can be a float number (such as "1.34"). Do not use any unit for the numerical data (don't use "1 cm"). It can also be a String (such as "male"). Data can be used as numerical data or as categorical data, see further below.

   It is best to prepare a fully complete data set, where each line has data for all parameters. FCS data files can't have empty parameters, so missing data will be replaced with the value 0 during conversion. Please be aware of that, as it might have an impact on the data anylsis in flow cytometry software!

2. The first line defines the number of parameters for the data file. For example, if the first line has 6 values and all following lines have 7 values, Data2FCS will only convert the first 6 values for each event and drop the 7th.

3. The first line may be used to define the name for the parameter. You may also specify the name with Data2FCS, see below.

4. When double-clicking Data2FCS.jar, a file selection dialog will pop up. Select your ascii data file to continue.

5. You can select various options for the conversion. Select the correct options so that the values in the table below are shown correctly.

   • "Use first line as parameter name": select this option if the first line of your ascii file lists names for the parameters instead of an actual event.
   • "Parameter separator:" select the delimiter that separated the values of each event. TAB means the tabulator, a common choice. Other options are an empty SPACE, the "," or the ";".
   • "Decimal point indicator:" are your values written with a period (1.23) or with a comma (1,23) as decimal point?

6. The table displays each event in a separete row and each value as a column. The header of the column indicates the name of the parameter, as defined in the ascii file. It can be simply "Param #1", etc, if there is no header line in the ascii file. If a header line is defined, then this column title reflects the labels used in the header line.

7. The first rows in the table allow conversion options for each parameter:

   • [Name]: enter the name of the parameter. If a header line is defined in the ascii file, the header is also placed into the [Name] field. However, once you manually edit the name (by typing into it), that value remains stable. Please always press the ENTER key when you enter a name, so that Data2FCS realized that you changed something.
   • [Log Display]: defines that the parameter shall be displayed on a log scale in the flow cytometry software. This option only defines the way how data is displayed. It does not transform the values! All values are stored as defined in the ascii file as float data.
   • [Categorical]: shall Data2FCS treat that parameter as categoricla data? An example would be a paramter for "male" and "female". This could be simply string values, or numerical values that indicate a category, such as "1" and "2". If the data is treated as categorical data, Data2FCS converts the data: The total number of categories in the parameter is screened. Then, the categorical values are sorted alphabetically. Then, the categories are stored as their index * 100. All this is necessary, as flow cytometry software can not have string values. Also, extremely low values, such as categorical counts, can cause problems - so they are multiplied by 100. So let's say you have a category "Gender" with the values "m", "f", "u", for male, female and unknown. The categories will be sorted to f-m-u, then the values will be changed to 100, 200 and 300. The value range will be set to 400 (see below).
   • [Jitter]: for categorical data, a jitter can be defined. The reason for this: as categorical data is simply 100, 200, 300, etc, displaying the values in flow cytometry software is difficult - you would simply see one thin line or one single point in the plot. Hence, a jitter can be applied that randomly distributes the events around the base-value of the category. This makes them more visible in the plots.
   • [Range]: Which range shall be used for displaying the data? Data2FCS by default picks a range that is the next-higher full number. You can enter your own range here. However, you can not enter a range that is lower than the default range. The "Range" value is ignored for categorical data.
   • [Highest Value]: For your information, Data2FCS shows here the highest value of the parameter in your data set.
   • [Skip]: Select this, if Data2FCS should skip that column for conversion.
   • [Status]: for your information, Data2FCS displays a status on your data set. "complete" means numerical values are available for all events. "incomplete" means that there are numerical values, but some events do not have a value. Please note that empty values will be replaced with "0" during conversion! "not numerical, complete" means that the parameter includes string values for at least one event. Maybe this is a category (see above)? Please note that Data2FCS replaces all non-numerical values with "0", if the parameter is not defined to be "categorical"! Finally, "not numerical, incomplete" means that there is at least one not-numerical value and at least one empty value in the data set of this parameter. Please note that these values might get replaced with "0" during conversion. In general, you should attempt to clean up your data file and to not have empty data points before conversion.
   • all following rows display the data in your file. Please note that "#missing#" indicates empty fields. Try to clean up all empty values before conversion!

8. After you selected all options appropriately, click on "Convert". Data2FCS will allow you to enter a file name for the converted data. Do not use your ascii source file as the destination file here!

9. If all goes well, Data2FCS will tell you in a little message. Now you can analyze your data in flow cytometry software.