Responses to Flow rate question

Rosson, Dan RossonD at MLHS.ORG
Mon Jun 18 08:00:56 EST 2001


	In the spirit of sharing all the perspicuity offered by the 2160
denizens of the Purdue cytometry mailing list, I am relaying the answers I
received to what I thought would be the "elementary" question of whether the
sample concentration ( hence the count rate) affects the data one obtains
during a FACScan run. I had expected to get several definitive answers from
my more experience brethren. What I found out is I'm less of a tenderfoot
than I thought. Therefore, I feel confident enough to state that I
personally find Calmain Prussin's and Paul Fallon's answers to be the most
cogent and Hank Pletcher's last comment about "three different flow
cytometrists" the most apposite. My surmise of the situation is that there
is indeed something in the circuitry that detects and eliminates cell
signals that are too close to each other. However, if the cells are really,
really, really close to each other, the circuitry will think two cells are
one and the signals will register as one just as two cells stuck together
register as one and we all know we have to turn on our doublet
discriminators when we do cell cycle analysis. If true, this would imply
that when we compare antigen levels between samples, like before and after
treatment, we should be careful to suspend and hence run the cells at the
same concentration, lest we get artifactually high MFI's for the sample that
is the most concentrated. Argh! something else to be careful about.
	So here they are:

____

Hi Dan,
I have a FACScan also, and my understanding is that if there are too
many cells for the machine to gather all the information on, a
percentage of cells will be ignored and their data will not be included
in the events you've collected (eg, more than 1 cell passes through the
flow cell in the path of the laser simultaneously).  You can slow the
flow rate to low on the control switch, and/or dilute the sample a
little.  I try not to have the cells more concentrated than a couple
million per ml.  I run a machine for research, not a clinical one -- I
do not know if the clinical protocols allow you to do this.
Good luck!

Beverly Barton, Ph.D.
Assistant Professor
Department of Surgery
UMDNJ-New Jersey Medical School


PS--Call BDIS and ask tech support--they will have the definitive answers!
Best,
Beverly
-------

Increasing the flow rate increases the frequency of doublets (2 cells) being
detected  as single events and thus yielding false double positive cells.

A couple of years back we tried running our FACSCalibur at rates over 3,000
events per second in order acquire sufficient number of events to detect Ag
specific cytokine responses. in a pilot, we stained separate samples for CD3
FITC and CD3 PE and then mixed the cells together after staining. Gating on
lymphocytes by scatter reduced the number of double positive cells, since
most of the doublets had a greater forward scatter. Even though we gated on
the lymphocyte cluster,  as  the rate increased, so did frequency of double
positive events. For the events we were studying, we found that 3,000 events
was the maximum event rate we could use and still be assured that our double
positive cells were actually not 2 signal positive cells triggering at the
same time.

Calman Prussin

-----

My vote is on ignore.  FACS user since 1983.

Deb

-----

I think the electronics detect coincident events and drop them from
analysis. The net effect is lower flow rate of events than reality. The
fluorescent measurements should not be effected. The was a posting in the
archives that measured the flow rate accuracies of the FACScan, but not the
fluorescence.
The fluid rate effects the sample core, so you will expect lower CV's on
the Lo flow rate.
-------

Hi Dan,

My understanding is that if another pulse (i.e. cell) arrives before
the instrument is finished processing the first one, it aborts both
events and waits for the next one to come through. You don't ever see
these abort events, but the cell sorters have counters that can keep
track of them.

Because cell sorters go much faster, and because we worry about
recovery, we keep track of these things. I seem to remember that the
deadtime, or processing time, of a FACScan is around 250usec. That's
why they recommend no more than 1500 events/sec as a trigger rate.
Newer FACSCaliburs have significantly shorter deadtimes (perhaps
50usec), and cell sorters are even faster (3-8usec).

As a result, going too fast just makes you less efficient, but
doesn't alter the data. It just means you don't see cells that go
through too close to each other. Of course, cells like to associate
with each other, so I suppose you could argue that there is a
selective cell loss.

BTW, you should know that if you ask three different flow
cytometrists a question, you'll get three different answers. I've
been asking folks at BD about system aborts for many years, and I
still don't know the whole story. I do have an old timing chart for
abort events on the old cell sorter, if you're interested.

Hank Pletcher

-----

I use a facsCalibur -
even for events within the range of tolerance for my machine I will
notice that at 20% max I will have lower CV than if I concentrated
the sample and ran it at 99% max thruput.

on all parameters.

-maciej

-----


test this on your machine by making a mixture of stained and unstained beads
at high concentration. I ran them up to 3000/sec and maintained low CV and
good S/N separation but you can easily determine that the electronics are
ignoring more and more particles as you go up in event rate.

-----
Hey Dan,

		I would think that there would be an optimal concentration
for efficiently running a sample on any given cytometer.  Too low of a cell
concentration and the event rate will be fast enough to have a unusable
signal to noise ratio.	Too high of a cell concentration and the cytometer
will not  only not miss events but will be unable to recognize individual
cells from doublets/groups of cells that will pass within the same time
window.  Addition of a piggyback cells which are singly positive in a given
channel while the first cell is positive in another will be recognized as
double positive.

		Good luck,


		Paul Fallon

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In addition to these replies, 9 people on the mailing list have their E-mail
auto replies on and hence informed me that they are out of town.





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