Re: Multiplus software
Kevin Becker - Phoenix Flow Systems (phnxflow@cts.com)
Thu, 20 Jul 1995 21:37:45 -0700 (PDT)
MultiPlus, as is most software for flow cytometry, is a very complicated
program with lots of features. MultiPlus is able to read in histogram or
listmode data from over 30 different file formats and then gate the data,
calculate a ratio between two parameters, generate dot plots, contour
plots, three dimensional views, perform a dual parameter subtraction,
compensation, align, move, annotate, smooth, overlay, Overton subtraction,
calculate statistics, store print files, write statistics to a data base
file, calculate up to three cell cycles, perform a background subtraction,
compensate for doublets, analyze a sub G0, G1 apoptotic peak, analyze a
standard peak, compensate for the difference in diameters of nuclei,
correct for the offset on your flow cytometer, and perform an error
analysis on the cell cycle results to evaluate the validity of the S-phase
to mention just the major features of MultiPlus.
Because MultiPlus has so many features and flow cytometry is such a small
market (few copies of any flow software product are sold compared to say a
word processor), testing all possible sequences of key strokes in
MultiPlus is a daunting job with limited resources at our disposal.
Phoenix Flow's policy is to fix any bugs in our software that can be
reproduced at Phoenix Flow. Sometimes this requires a customer to expend
some extra effort to document the exact key strokes so the problem can be
reproduced at Phoenix Flow. A statement like "setting a region in the dot
plot histogram and trying to analyze the DNA histogram.... the program
crashes and one has to reboot the computer." is an inadequate explanation
of when the software crashes.
Thank you Dr. Simms for providing the extra documentation as I requested in a
previous email to track down this nasty bug. We have now discovered the
bug!
On another issue, I would like to address the comments of Jenny Bryant on
MultiPlus and histogram subtraction. Ms. Bryant says, "When you use Mplus
to analyze two single parameter histograms, one negative control and one
test, and you select the subtract negative option, the result is not
always correct. MultiPlus appears to rescale the negative control
histogram to a scale unrelated to the real one and then it uses this
incorrect value in the subtraction process. The result is thus unusable."
What you have observed is the Overton method(see Cytometry 9:619, 1988) of
histogram subtraction. The object of Overton subtraction is to compare
DISTRIBUTION SHAPES (not number of cells) to determine what events of the
unknown data are positive. The method thus begins by normalizing the
control data to the same number of events as the unknown. The control
distribution is then subtracted from the unknown (positive stain) and the
number of events left over in the "positive" is the result used. Note
that the unknown, or "positive" histogram is itself never normalized.
Thank you and keep those comments flowing,
C. Kevin Becker
C. Kevin Becker
Phoenix Flow Systems, Inc.
CD ROM Vol 2 was produced by staff at the Purdue University Cytometry Laboratories
and distributed free of charge as an educational service to the cytometry community.
If you have any comments please direct them to Dr. J. Paul Robinson, Professor & Director, PUCL,
Purdue University, West Lafayette, IN 47907. Phone:(317) 494-0757; FAX (317) 494-0517; Web http://www.cyto.purdue.edu
EMAIL robinson@flowcyt.cyto.purdue.edu
