Major New Features In

FCS 3.0 is different from FCS 2.0 in the following ways.

1. The FCS 3.0 TEXT segment may be split into primary and supplemental TEXT segments. The primary TEXT segment must contain all required keyword-value pairs and be located entirely within the first 99,999,999 bytes of a data set. The supplemental TEXT segment can contain only optional keyword-value pairs and may be located anywhere in a data-set after the HEADER segment. The byte offsets to the primary TEXT segment are found in the HEADER segment. The byte offsets to the supplemental text segment are found in keyword-value pairs in the primary TEXT segment.

2. The HEADER has been modified to accommodate data sets longer than 99,999,999 bytes. A data-offset value that requires more than 8 bytes is now represented by placing a "0" in the HEADER for both the begin and end values of the DATA segment. The actual byteoffset values are found in the primary TEXT segment of the data set. The new keywords that describe offset values are $BEGINSTEXT and $ENDSTEXT for the byte offsets to the beginning and end of the optional supplemental TEXT segment, $BEGINDATA and $ENDDATA for the byte offsets to the beginning and end of the DATA segment, and $BEGINANA-LYSIS and $ENDANALYSIS for the byte offsets to the beginning and end of the optional ANALYSIS segment. This system allows the vast majority of data files to be backwards compatible with analysis software designed for previous FCS versions. However, a "0" byte offset in the HEADER will prevent these older versions of analysis software from reading very large data sets, avoiding read errors or partial data reads.

3. An optional 16-bit Cyclic Redundancy Check (CRC) word has been added to the end of each data set. This internal check word allows for data-set integrity checks (Press et al., 1992).

4. To enable third-party or off-line analysis software to read and interpret data correctly, the keyword $PnE is now required for each parameter. The $PnE keyword describes the method of amplification used for a given parameter using two floating point values f1 and f2. With logarithmic amplification, f1 specifies the number of logarithmic decades and f2 gives the linear value that would have been obtained for a signal with a log value of 0. When linear amplification is used, f1 and f2 are set to 0. For example, "/$P1E/3,0.1/" specifies that the data for parameter 1 were collected using a three-decade logarithmic amplifier and the 0 channel is equivalent to the linear value 0.1.

5. The new keyword $COMP has replaced $DFCiTOj to describe the amount of fluorescence compensation employed in the collection of the data. The keyword value of $COMP represents a compensation matrix. The matrix has n rows and n columns, where n is the number of parameters. Both positive and negative values are allowed. A positive or unsigned value indicates additive compensation, while a negative value indicates the more common case of subtractive compensation. The elements are stored in row-major order, i.e., the elements in the first row appear first. For example, with a four-parameter file, matrix element seven of sixteen total elements (which would fall in row 2, column 3) indicates the percentage of parameter 3 that has been electronically subtracted from or added to parameter 2.

6. There are a number of other new optional FCS TEXT Segment keywords. $CSMODE, $CSTOT, $CSVBITS, and $CSVnFLAG specify added parameters to identify cell subsets. $CYTSN specifies the cytometer serial number. $TIMESTEP has been added to more accurately define how a TIME parameter is measured. The following optional ANALYSIS segment keywords have been added to enable specification of cell subsets: $CSDATE, $CSDEFFILE, $CSEXP, $CSnN, and $CSnNUM.

7. $UNICODE enables the specification of certain keywords in languages that cannot be represented in ASCII. UNICODE is an international standard that enables computer representation of most of the world's languages (Unicode Consortium, 1991). The characters for each language are represented as 2-byte codes. The first byte represents the UNICODE "page" on which the character is found and the second byte represents the individual character. There are 65536 characters available.

8. The keyword values representing a year are increased by two bytes to include a century designation (e.g., 1996 rather than 96) for the keywords that represent a date ($DATE and $CSDATE).

9. The $PnG keyword has been added, describing the linear gain applied to a signal.

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