The FCSExtract application was developed to be a small utility that will allow you to extract Flow Cytometry Standard (FCS) data from a binary FCS file and convert it to an ASCII text format, which can be read by a variety of software analysis packages. An example of working with this data in R is provided.
You may be frustrated if you attempt to analyze your flow cytometery data in a FCS file using any third-party software analysis tool. The FCS file is a binary file, and contains a number of non-ASCII text characters, which are unprintable, and unreadable by many programs. You cannot just load a FCS file into Excel or other analysis tool easily.
FCSExtract is a small application, approximately 8Kb, that extracts raw flow cytometry data from a FCS binary file and converts the data to ASCII text for use by a variety of software programs, including R.
The tool will automatically extract the following types of data from a FCS file, which must be extracted in this order.
The format of the resulting ASCII file depends on the target software. The specific file format for R is described in the Example section below.
Raw Flow Cytometry Data (DC, CD4, CD8, CD16, CD19, CD34)
Values and percentages for each of the above markers, including positive/negative, or intensity, for every cell in the sample.
Any metadata associated with the sample.
If there is a possibility of your sample containing cells with very large or non-integer values, then the Large Cell Viability Check (L-CVH) must be performed prior to exporting data to a text file. To find this option, click on the Settings button in the main window of the tool. The L-CVH screen appears, where you may indicate that it is optional, and the check itself will appear as a condition of the export.
If the L-CVH process finds a large or non-integer value (e.g. value > 10,000) in any cell of the sample, the tool will stop the export process, and will not attempt to process the remaining data.
The tool extracts each of these data types in the order listed above, then generates a standardized text file for each object that is created, and then aggregates all of these files together into a single FCS file that can be imported into R or other analysis software.
The tool allows you to extract any information from your sample including:
Objects (cells, clumps, etc.)
Markers (SIG, FITC, PE, APC, etc.)
Groups (gated populations)
Phenotypes (CIT, POS, BRL, FSC, SSC, etc.)
Flags (such as artifact, dead, alive, etc.)
You may enter the following values in the FCS file to indicate what data type should be imported from the FCS file and then converted to text. This tool will interpret each of these values as one of the following strings.
“raw” indicates the
FCSExtract.exe is a Windows 95/98/Me/NT/2000/XP command line application that extracts FCS data from a binary FCS file. It writes a text ASCII formatted FCS file. The ASCII file is designed to be read by any FCS analysis software and any spreadsheet application that can read text files, or be read by R.
FCSExtract allows the user to modify the output FCS file as desired. It is intended for exporting data from a Flow Cytometry Software Analysis System (FCSAS) or other data analysis tool.
FCSExtract has no external dependencies; it does not need access to any files on the user’s system. It can work with data from any source – binary or ASCII text format.
Your FCS file should be a binary format file, with ASCII characters from 0-127, no embedded binary data, no non-ASCII characters. If you don’t know how to convert an FCS file to a binary file, you can use another binary to ASCII conversion tool like BinConvert.
You may use this program with an 8-bit or 16-bit data file, including compressed (zipped) data. FCSExtract can extract data from FCS files with either 4- or 8-bit floating point data. The 8-bit floating point data can be obtained from an FCS file that contains ASCII characters 0-255.
The supported data format for the extracted data is as follows:
0,$hFFEE,$t0,$d0,$s0,$S0,$s0 is the file name,
1,$hFFEE,$t0,$d0,$s0,$S0,$s0 is the label name,
2,$hFFEE,$t0,$d0,$s0,$S0,$s0 is the memory,
3,$hFFEE,$t0,$d0,$s0,$S0,$s0 is the channel,
4,$hFFEE,$t0,$d0,$s0,$S0,$s0 is the data type,
The goal of the FCS Extract tool is to extract the raw data contained in an FCS file, and reformat it into a more readable ASCII file. The extracted raw data can be exported to a text file, and read into a spreadsheet application like Microsoft Excel or GNU Octave for analysis.
In most cases, the raw data in the FCS file contains information on cell size and intensity. The cell size information is typically stored in fields named “Wx” or “Log10(Wx)”. The intensity information is usually stored as the fractional total value in a field named “Log10(Total)”.
Why Convert FCS Files?
As cell size data is one of the most important information contained in an FCS file, a lot of effort has gone into developing software tools that can analyze FCS files. The only problem is, most existing analysis tools have to be configured to understand these files, and converting a binary file to a text file is not usually a trivial task.
What is FCSExtract?
The FCS Extract tool solves this problem by taking an FCS file and reformatting it into an ASCII format, which can be read by most software programs. This is done through a series of text extraction steps. FCS files can be extracted from a number of file formats, including:
When you use the FCS Extract tool, it will extract the data from the file into two text files, a raw data file, and a text file containing all the data extracted from the FCS file. These can be exported to a spreadsheet application like Microsoft Excel or GNU Octave for analysis.
Using FCSExtract to Extract Data From FCS Files
The FCS Extract application is simple to use. Just open the file, select the data types to extract, and click Extract.
The FCS Extract tool will then display the raw data extracted from the FCS file, and the text file containing all the extracted data. You can extract data from any of the files that are supported by the FCS Extract tool. When extracting from a FCS file, the raw data will be extracted from the FCS file into a text file. This text file will then be imported into a spreadsheet application such as Microsoft Excel. The text file containing the extracted data is a formatted ASCII file, which can be read by a number
This application will convert a.fcsextract file to a.txt file format that can be read by FlowJo or Summit. This will allow you to analyze your data using the combined software packages.
There is currently no version number available.
Must be installed on a system with the FCS File extension installed.
This application is only packaged as a.exe file, which must be run directly from the hard drive. There is no installer available at this time.
Using the FCSExtract utility:
Extract the file
Choose the output file path from the list
Choose the desired output format, either.txt or.xls. Click the “Extract” button, and the.txt or.xls file will be saved to the specified path.
Output files are written in UTF-8 character encoding, and the.txt and.xls files have the same encodings.
Programming languages used:
Visit the following web site for information about FlowJo and Summit software, and more information about FCS files.
If you have questions or would like to report a problem with this utility, please use the following e-mail address.
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