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In order to calculate the S phase fraction in a cell cycle correctly, the
backround debris has to be taken into account because many times the
backround not only is found to the left of the first G1 population but
also extends into the S phase. This is especially true in paraffin fixed
samples where there is lots of debris. In fresh samples,
because the backround is much lower, people often choose to ignore the
debris and gate it out when they are collecting the dna histogram.
However, if you think about it the disaggregation of any tissue can
generate debris or there is always junk floating around in cultured
cells. For this reason and because of the need for general rules to
standardize DNA analysis, we say always use backround subtraction in cell
cycle analysis. The backround subtraction option can be found in
MultiCycle on the menu page right after you have selected the number of
cell cycles.
Because the setting of a doublet gate is such a subjective thing,
the DNA Consensus (see Cytometry 14;472,1993) came up with a new
measurement of the amount of backround in a sample. This is called
B.A.D. which stands for Backround Aggregates and Debris. This means the
amount of backround, aggregates and debris under the cell cycle from
the first G0/G1 peak to the last aneuploid G2 peak. However, to
calculate B.A.D. your cell cycle analysis software must also be able to
calculate the percentage aggregates in the DNA histogram which is another
whole can of worms in itself. Presently, the only cell cycle softwares
that calculate B.A.D. are produced by the small software houses, not the
machine manufactures.
The point is, for any cell cycle analysis software to calculate
the percent backround or the percent B.A.D., it needs some of the
information to the left of the histogram, the more the better. (Too be
continued)
C. Kevin Becker
Phoenix Flow Systems, Inc.
San Diego, CA
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