[Cytometry] Problem with BD CS&T laser settings

Ian Dimmick ian.dimmick at newcastle.ac.uk
Wed Jul 22 13:45:57 EDT 2009


I would like to add to this discussion , as a user who has used the CS&T bead set up on all of my instruments for the past year , I find the QC protocol attributed to these beads to be very informative and enables things like adjustment of laser delays to be much more standardised, the linearity check to be much less hassle and overall to be a very nice overall QC package . We run these beads each day for each instrument , however this is done for our end users, we do not ask users to do this as we feel we want to know the status and interpret with appropriate action as a core facility . The FACSAria we have is a 4 laser system (SORP) and because we run 5 different pressures on the sorter so we therefore  run 5 different CS&T protocols (70 micron / 70 PSI, 70 micron / 35 PSI, 85 micron /40 PSI, 100 micron .20 PSI and 130 micron /14 PSI). All 5 pressures will require 5 sets of lasers delays that will differ from one a nother , The LSR II and FACSCanto II has only one configuration as there is only one pressure involved when running these instruments. If you look at the history of the laser delays in the CS&T log file you will see that the laser  position/sheath pressure/direction of wind does vary from day to day , therefore fluorescence intensities will be affected and so compensation values, so justifying running the beads on a day to day basis .
I would also like to add that we run these beads through DIVA software  , Cellquest, Flomax  any other instrument available to us  to enable us to visualise the discrimination between the different levels of fluorescence for any laser and or detector, this gives us useful initial data , very easily , especially the discrimination between the dim and mid bead for the detectors. The note of warning I would give is that if the nozzle on the aria is seated slightly incorrectly , or is very slightly blocked the CS&T may still run and give you misleading laser delays that are only correct for the temporary glitch , therefore it is always useful to look at the laser delay history .In addition I feel that the 50 volt tolerance is too wide and suggest a 10 volt tolerance .
Ian Dimmick
Flow Cytometry Core Facility Manager
Institute of Human Genetics
Bioscience Centre
International Centre for life
Newcastle Upon Tyne
NE1 3BZ
UK
Ian.Dimmick at ncl.ac.uk
Tel 0044 191 2418831
Fax 0044 191 2418666
(mob) 0044 7970344823
http://www.ncl.ac.uk/ihg
________________________________________
From: cytometry-bounces at lists.purdue.edu [cytometry-bounces at lists.purdue.edu] On Behalf Of McClellan, Steve [smcclell at ufl.edu]
Sent: 22 July 2009 17:23
To: Kruger Gray, Huw; 'cytometry at lists.purdue.edu'
Cc: 'Charles_VanFossen at bd.com'
Subject: Re: [Cytometry] Problem with BD CS&T laser settings

Hi Zip,
You do indeed need to make a separate config for each different nozzle and baseline it.  Changing nozzles requires you to go into CS&T and choose the appropriate config, but if your last performance check is current, you do not need to re-run one, it will use the settings of the last performance check that passed.
Kind regards,
Steve

Steve McClellan
Sr. Biological Scientist
Interdiciplinary Center for Biotechnology Research(ICBR)-
Flow Cytometry Core Laboratory
University of Florida
352-273-8185 (office)
352-273-8070 (fax)
smcclell at biotech.ufl.edu
Cancer/Genetics Research Complex-Room 292A

-----Original Message-----
From: cytometry-bounces at lists.purdue.edu [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Kruger Gray, Huw
Sent: Wednesday, July 22, 2009 10:15 AM
To: 'cytometry at lists.purdue.edu'
Cc: 'Charles_VanFossen at bd.com'
Subject: Re: [Cytometry] Problem with BD CS&T laser settings

Greetings,

     Indeed, we also have had some issues here with the CST beads. For instance, when we tried to run CST after exchanging our usual 70um. nozzle for the 130um. one, CST set the laser delays incorrectly and these then had to be re-set manually. Eventually, we traced thus problem down to the fact that we still had been using the original configuration file for the 70um. nozzle and should have created one specifically for the 130um. nozzle. I am not sure yet if we then would have to run a new base-line for the substitute configuration, or if we just could run a performance check, but I suspect the former to be the case. Also, because we had been using the original configuration with the larger nozzle, when ever we stopped the stream for any reason, when we then re-started it, the system re-set the sheath pressure back to the original setting used with the 70um. nozzle, ie. 70 psi. We never allow any of our users to run CST, by the way, but only the facility's staff and we alwa!

 ys instruct users to accept the "Use CST settings" option.

Good luck,

Zip.

Huw S. ("Zip") Kruger Gray, Ph.D.
>>>--->

Director, Flow Cytometry Core Facility,
Sylvester Comprehensive Cancer Centre,
Miller School of Medicine,
University of Miami.
1600 NW 10th Ave., RMSB 3061
Miami, FL 33136.
305-243-5019 (Office),
305-213-3325 (Mobile).
www.sylvester.org/flowcytometry<http://www.sylvester.org/flowcytometry>


--- On Tue, 7/21/09, Christopher Bare <flowmail at verizon.net> wrote:

From: Christopher Bare <flowmail at verizon.net>
Subject: Re: [Cytometry] Problem with BD CS&T laser settings
To: "'Felix Heymann'" <felix.reichel at ukb.uni-bonn.de>
Cc: "'Cytometry List'" <cytometry at lists.purdue.edu>
Date: Tuesday, July 21, 2009, 2:46 PM
Felix,

You are welcome, and you are correct!

We run UltraRaindows in addition to CS&T by applying what BD calls
"Application Settings." This changes our voltages by an amount relevant to
the CS&T defined voltages to bring our beads back to the consistent channel.

Again, be warned that Application Settings will change your Area Scalings IF
when you set the Application Settings, your scalings are DIFFERENT from the
CS&T at that moment. (It's all about the DeltaFromCS&T). So again, be
careful defining the Settings.

Our best typical approach has been to instruct every user to "Use CS&T" but
"Duplicate Without Data" to preserve their PMT voltages. So far, that
sequence works.

Cheers!

-cbb

> -----Original Message-----
> From: Felix Heymann [mailto:felix.reichel at ukb.uni-bonn.de</mc/compose?to=felix.reichel at ukb.uni-bonn.de>]
> Sent: Tuesday, July 21, 2009 12:35 PM
> To: Christopher Bare
> Cc: 'Cytometry List'
> Subject: Re: [Cytometry] Problem with BD CS&T laser settings
>
> Hi Chris, hi all,
> thank you for this enlightening description, I think, it is very
> important that people doing flow on the BD machines know about these
> pitfalls, it can not be stressed enough that there is probably very
> little that can mess up your experiments more than wrong laser settings.
> Anyway, I still have my issues using the CS&T do do a full setup of my
> instrument, since (as I mentioned earlier) at least for me the CS&T
> settings tend to be total crap in terms of the PMT voltage settings and
> I normally have to readjust them anyway (big time!) before my actual
> measurement.
>
> I actually reran the CS&Ts together with CaliBRITE and Rainbows on our
> Aria II for the configurations, doing a performance check first (which
> was ok), but when I actually ran the beads as a standard sample, quite a
> lot of PMTs were way out of line and set either too low (no good
> discrimination of dim and med CS&Ts) or too high (bright beads crammed
> to the highest channels). This is in fact a problem, since I can not
> teach the flow users here to rely on the CS&T settings, because they are
> utter nonsense in terms of the PMTs, although area scaling, laser delay
> and window extensions are adjusted accurately.
>
> So what would be a good way to solve this issue? My idea so far would be
> to tell the people to stick to the 'use CS&T' in the dialogue when asked
> to have the delay etc adjusted right but disregard the PMT voltages and
> start with their single stains from scratch to adjust the PMTs? I have
> no other idea how to do it so far, so if you come up with a different
> solution, please let me know.
>
> I really like the idea behind the CS&Ts, but either I am hopeless using
> them or there may be some room for improvement from BD side to narrow
> the gap between artifically labled, small and very bright beads compared
> to big unregularly shaped cells with highly variable fluorescence
> properties.
>
> Best regards
>
> Felix
>
> Felix Heymann
> Scientific research fellow
> Medical clinic III, University hospital Aachen
> Germany
>
>
> > Flownauts!
> >
> > We at Rockefeller recently discovered a dramatic "feature" of the DiVa 6
> > software utilizing CS&T to set laser delay and area scaling when applied
in
> > a multi-account (multi-user with different logins) setting that can
cause
> > drastically wrong values to be applied. That, of course, can make your
> > parameters falsely negative for the entire octagon/trigon.
> >
> > The Problem:
> > When a user logs in, DiVa checks the database against the Current CS&T
> > values stored on disk. If there is a mismatch, the user is given a
Dialogue
> > Box asking to "Use CS&T" or "Keep Current." If the user clicks "Keep
> > Current," the instrument laser settings are changed to non-current
> > (out-of-date, invalid) values EVEN IF the user does NOT have permissions
as
> > assigned in the "Administration" window.
> >
> > The laser settings that the user previously used may be very very VERY
> > different from the current instrument configuration (say, if a new laser
was
> > added).
> >
> > A Solution?
> > If an account does not have permission to adjust delay and scaling, they
> > shouldn't get the Dialogue Box asking them to keep current! BD should
> > reprogram DiVa to ALWAYS apply Current CS&T settings for
non-permissioned
> > accounts.
> >
> > We have instructed our users to ALWAYS choose "Use CS&T," but not
everyone
> > listens, or understands. Failure can render subsequently acquired data
> > invalid and useless. And unlike compensation or airbubbles, this can not
be
> > retrospectively repaired.
> >
> > A fix is better than a workaround.
> >
> > I found this because a user complained that his CD3 APC was all a big
> > negative smear. I noticed his 640nm delay was 18 instead of 32. I
applied
> > Current CS&T and his positive population magically appeared.
> >
> > We run two self-service LSRII (one 4 laser, one now 5 laser). We define
a
> > single Instrument Configuration for all (200+) users with parameters
names
> > for laser source and PMT position (e.g. 488-B) in order to prevent any
> > conflicts for mismatched configs and avoid end-user mistakes. We deny
users
> > the ability to change delay and scaling, instead administratively run
CS&T
> > weekly and post the numbers on the machine for user confirmation. We do
not
> > allow users to run CS&T themselves, or create and use alternate
Instrument
> > Configurations. We recently upgraded one machine from 4 to 5 lasers, and
> > reordered their intercepts. This changed all delays (except 488).
> >
> > The way DiVa sets a user's laser delays and scalings:
> > If the user chooses "Keep Current," DiVa sets the values according to
the
> > Last Tube created by the user during the previous login. If the user
does
> > not have any tubes in the database, DiVa uses the user values from a
> > different user_account_laser_defaults table, which is set when the user
last
> > logged OUT. So if CS&T has been run since the user last logged in, there
> > will be a mismatch and the user gets the Dialogue Box.
> >
> > Sincerely,
> > Cris Bare
> > Rockefeller University
> > Flow Cytometry Resource Center
> >
> >
> > _______________________________________________
> > Cytometry mailing list
> > Cytometry at lists.purdue.edu</mc/compose?to=Cytometry at lists.purdue.edu>
> > https://lists.purdue.edu/mailman/listinfo/cytometry
> >
> >

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