Appropriate number of events to collect for 3 color flow cytometry ?
Cindy Zuleger
clz at medicine.wisc.edu
Thu Jan 17 18:54:06 EST 2008
Dear Abdul-
I have copied and pasted an email Mario Roederer sent out a few weeks ago addressing the
interpretation of small populations. I think his comments may address your question
regarding # of events to collect.
Cindy
Email from Mario Roederer:
Rick,
there is no "general cut off". Many people have wondered what to
interpret as "real"... but the bottom line is that every single event
is "real"; the only question is whether it is biologically
meaningful. To assess that, one must do the same thing as we do for
every other kind of experiment -- to determine the range of values
that are measured experimentally.
Thus, if you have a particular staining panel, and in a particular
gate you find zero cells in all 50 controls, then finding even one
cell in that gate for your experimental sample becomes "real" and
biologically meaningful. On the other hand, if you find an average of
1000 events in this particular gate in all controls, and you find 950
events in your experimental sample, then the 950 events are still
real, but they don't comprise a difference from the control.
So there is no absolute threshold that is meaningful; it is different
for each experiment. If you have 0.05% in your gate, whether it's 2
cells or 2000 cells, I would want to see the same backup data to
demonstrate that this is "biologically meaningful".
Of course... the PRECISION of a measurement will be far different
depending on how many events are counted. If you count only 2 events,
then the precision is about +/- 70% (square root of 2 divided by 2).
If you count 1000 events, the precision is about +/- 3% (square root
of 1000 divided by 1000). So when you have few events, that doesn't
mean they aren't believable, it just means that you don't have as good
a handle on the true fraction that they represent as you would with
many more events. Note that the precision for a rare populations is
ONLY dependent on the number of events in that population that were
counted -- i.e., the precision on 100 events is the same whether the
overall sample had 100,000 events (0.1%) or 100 million events
(0.0001%)!
mr
On Dec 12, 2007, at 4:10 PM, <Rick.Schlichtemeier at UCHSC.edu>
<Rick.Schlichtemeier at UCHSC.edu
> wrote:
> Hi,
> I was wondering if anyone had any information as to how to interpret
> low numbers of cells of interest.
> Is there a general cut off and number of cells that is generally
> accepted? I have heard that it is okay
> to go down to 0.05 as long as 50 cells are counted. But I wanted
> to be sure about this.
> Thanks very much
> Rick Schlichtemeier
> Research assistant
> Univ of Colorado Health Sciences Center
>
>>> "Abdul Khan" <khan.abdulh at googlemail.com> 1/16/2008 5:52 PM >>>
Hello Again!
I am sorry for posting too many emails in the forum, can't get any better
advice than here.
I am just wondering and interested to know that if there is any particular
number of events (say 10,000 or 20,000) I need to collect while doing 3
color flow cytometry in whole blood.
Thanks for your help.
Best wishes,
Abdul
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