[Cytometry] Proper usage of CV in data analysis

Guy Hermans Guy.Hermans at ablynx.com
Mon May 16 06:16:00 EDT 2011


Hi V.,

Short answer: "no".
Long answer: "depends" ;-)

We routinely generate cell membrane marker transfected cell lines here, and single-cell clone from those lines by FACSsorting. We stain for the surface marker in question during the single-cell sorts. Transiently transfected cell line populations typically multi-log wide fluorescence histo's, either without prior antibiotic selection or after having been under such selection for a week or two. Single-cell cloning derived clones typically show much narrower peaks at various intensities (using same probe in same FACS experiment after growing out the clone). So it least versus transiently transfected cell populations, most (but not all) single-cell sorted clones indeed show a narrower distribution. Reasoning the reverse is pretty dangerous, though.

Of course, keep in mind these transfectants are usually driven from strong, constitutive promoters. If expression of your gene of interest were dependent on cell cycle, to name but one possibility, non-synchronized growing cell populations would seem to be highly heterogeneous at any point in time as well... even if cells at the point in cell cycle would express exactly the same number of molecules and so, in the end "It depends".

G

Guy Hermans 
Principal Scientist 

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-----Original Message-----
From: cytometry-bounces at lists.purdue.edu [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Vinko Tosevski
Sent: Friday, May 13, 2011 10:36 AM
To: cytometry at lists.purdue.edu
Subject: [Cytometry] Proper usage of CV in data analysis

Dear all,

The other day a colleague asked me if there's a way to assess the
clonality of her transfected cell population based on the width of the
peak she's seeing in the cytometer. While thinking about this, I came
up with some questions regarding the use of coefficient of variation
in flow cytometry for assessing the dispersion of the measured
population. I would appreciate if you could expand my perspective on
these topics...

Can I draw some conclusions about clonality of the population by just
looking at the width of the histogram peak? If yes, how and what's the
meaning of it? What do I compare it with? I assume comparing it to the
width of untransfected cells makes no sense since those ones are not
carrying the "trait" that I am looking at to begin with (not
expressing fluorochrome - tdTomato, by the way). How does one approach
this?

Looking at the peak of untransfected cells made me ask myself another
question - are CV values properly used in addressing the dispersion of
the measured fluorescent populations? I mean, those are not normally
distributed populations (they are log-normal at best) so use of CV
would be inappropriate since assumptions of normality are not met. I
guess robust CV would be the way to go, or...? But, in addition, does
any type of CV makes sense?? If I remember my textbooks correctly, CV
as a measure of dispersion only makes sense if one is dealing with
measurements of a ratio scale (in sense of statistics). Could
fluorescent measurements be actually seen as ratio scale? To me, it
sounds more like interval scale, but am I right, then, to conclude
that CV should not be used at all?!? At the moment I suggested her to
use interquartile range as a measurement, but I am really looking
forward to hearing some thoughts on this.

I appreciate your input!

Best,
Vinko


--
Vinko Tosevski
Institute of Experimental Immunology
University of Zurich, Irchel campus
Building 44, Room J42
Winterthurerstrasse 190
CH-8057 Zurich
Tel:  +41 44 6353706
Fax: +41 44 6356883
tosevski at immunology.uzh.ch
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