RE: query on LPAs: replacing tritiated thymidine with flow

From: VSH - Tech Support (tech@vsh.com)
Date: Tue Aug 13 2002 - 13:45:10 EST


Alice is correct that the ModFit LT Proliferation Wizard is designed to work
with indistinct populations frequently seen when doing PKH or CFSE analysis.
The theoretical positions she describes are the calculated mean position of
the daughter populations. These positions are calculated using information
about the log decades in your system. By default this calculation is based
on 4-log decades which gives a theoretical position shift of -19.19 channels
for each daughter population. Changing the default setting is as simple as
entering the actual log decades of your instrument. The software then
adjusts all the calculations for the daughter populations.

ModFit LT also has the capability of automatically calculating asymmetric
cytokinesis. This function is accessed through the Proliferation Wizard.

In some cases histograms clearly show an asymmetrical pattern with respect
to the shift of the daughter populations. We know that the CFSE samples can
be affected by auto-fluorescence which shifts weakly fluorescing daughter
populations upscale. We also know the analysis can be affected by the
characteristics of the log amplifier. If the log amplifier has
irregularities in its signal response it could result in irregular shifts in
daughter populations. The only way to truly know the cause of these shifts
is to perform a careful analysis of the log amplifier's characteristics.
Once these characteristics are known the standard proliferation model can be
adjusted.

There have also been several occasions where customers have supplied data
showing distinct PKH and CFSE populations. Using the flexibility of ModFit
LT's model editor we were able to provide these customers with models that
automatically adjusted for the asymmetrical nature of these histograms.

As many of our customers will attest, we are glad to assist any ModFit LT
user with these special needs.



Best Regards

 Don

Donald J. Herbert
Technical Support Manager
Verity Software House, Inc.
PO Box 247
45A Augusta Road
Topsham, ME, USA  04086

Phone: (207) 729-6767 ext.190
Fax:   (207) 729-5443
email:  tech@vsh.com
web: www.vsh.com





-----Original Message-----
From: Alice L. Givan [mailto:Alice.L.Givan@dartmouth.edu]
Sent: Friday, August 09, 2002 9:36 AM
To: cyto-inbox
Subject: Re: query on LPAs: replacing tritiated thymidine with flow



Flowers,
ModFit software from Verity has  a "Proliferation Wizard" that does all the
proliferation
calculations for you from cells stained with CFSE or a PKH dye  (for
example,  it
gives you the precursor frequencies of the proliferating cells and it also
gives you
a proliferation index that tells you how many more cells you have now than
when you
started the culture).

What ModFit does is model the intensity histogram of the cells --- so it can
use the
separate or quasi-separate peaks found in some cases with CFSE-stained
cells.  Or it
models the theoretical positions of the peaks for dividing cells if you are
staining
with the PKH dyes (that don't give such good intensity separation) or if
your CFSE
staining hasn't given separate peaks.

Main problem with ModFit software is that it is using the theoretical
position for
the intensity peaks of the proliferating cells -- it won't be as accurate if
cells
are dividing asymmetrically and the proliferation peaks don't match the
theoretical
positions expected for halving of intensity with each division.  We have
found that
it matches the peaks quite well with CFSE -- as long as you use a
correction for the
particular amplifcation factor for your  log amplifier (the log decades full
scale
are hardly ever exactly 4).  And we have also found that the ModFit software
gave us
values that correlated well with the known values of a model system.

Conflict of interest:  After writing our paper on using PKH and flow to
quantitate
precursor frequencies of antigen-specific T cells (J. Immunol. Methods 230:
99, 1999),
I worked with Verity to get them to include the precursor frequency
calculation in
their ModFit software.  I have no financial interest at all in this --- but
am somewhat
devoted to the concept of using CFSE or PKH to do precursor frequency
calculations
as I feel this method provides much more complete analysis of proliferative
responses
than does tritiated thymidine (which is only a bulk assay related to the
total number
of proliferating cells at the time of the assay).

Others who,  before us, have used CFSE and flow to quantitate precursor
frequencies
are Wells et al (J. Clin. Invest. 100: 3173, 1997) and Song et al (J.
Immunol. 162:
2467, 1999).  If I am missing any references on this,  please let me know.

Alice

Alice L. Givan
Englert Cell Analysis Laboratory
of the Norris Cotton Cancer Center
Dartmouth Medical School
Lebanon, New Hampshire NH 03756
tel 603-650-7661
fax 603-650-6130
givan@dartmouth.edu



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