[Cytometry] Problem with BD CS&T laser settings

Uriel TK utk at urieltk.com
Wed Jul 22 17:32:19 EDT 2009


Hi there,
It would seem there is some confusion regarding the role and objective of the CS&T procedure and beads. I actually discussed this with Joe Trotter himself in the last ISAC and he was kind enough to enlighten me. I'll try to be half as didactic as he was!

The CS&T is mean to follow up the cytometer's performance and set its "hardware" parameters.

By setting its hardware parameters I mean the area scaling and laser delays. Basically, finding the optimal settings for the hardware.

By following up the cytometer's performance I mean make sure that it stays relatively constant over time, and detect any significant shifts which could indicate problems. How do you do that? How do you follow up a machine's performance? With a standard. Since flow cytometers don't have an internal standard, you use beads - an external standard. These beads have been tested lot by lot and their parameters are known. Then you use that external standard as a scale to measure your machine. By tracking the optimal voltages and CVs etc that the machine produces for the beads you can see if the machine is drifting or shifting or something happened, since you assume the beads themselves, the standard, don't change. Thus the parameters obtained when doing the CS&T as optimal are optimal FOR THE BEADS. And you hope that that optimal range stays constant i.e. the machine stays constant.

Thus, with CS&T you find the best hardware parameters that should be used for the machine, and using those settings you determine whether the machine is keeping its performance evenly. It follows from this that the hardware settings should be kept as determined by CS&T, while the optimal settings for the beads, the voltages of the detectors, are only a follow up measure of the performance and are not meant to be the "best settings to use for all the applications". Indeed, a whole tutorial and a couple of posters in the last ISAC was dedicated to show the fact that careful and correct voltage setups are essential to achieve the best sensitivity for your given application, and how to find that. 

The bottom line is, use CS&T to track your machine and make sure it is behaving well, apply the laser delay and scaling etc according to the CS&T settings, and let the users set the voltages, as they should, according to their needs. Hopefully they know how to set them right. 

An exception: for people using doublet discrimination with FSC A vs H, sometimes it is needed to change the FSC area scaling to have them match in a useful way. After discussing this with our core manager, he gave me the required permissions after verifying that I knew what I was doing. In any case we are instructed to ALWAYS choose "use CS&T settings" and then copy the voltages as needed from previous experiments. I would recommend copying them by hand and not with copy/paste to avoid inadvertent copying of compensations.

Best regards,
Uriel.

Uriel Trahtemberg, M.D. M.Sc.
PhD student
The Laboratory for Cellular and Molecular Immunology
The Hebrew University - Hadassah Medical Organization
Jerusalem - ISRAEL

“Be careful of reading health books, you might die of a misprint.”
	Mark Twain

> -----Original Message-----
> From: cytometry-bounces at lists.purdue.edu [mailto:cytometry-
> bounces at lists.purdue.edu] On Behalf Of Christopher Bare
> Sent: Tuesday, July 21, 2009 9:40 PM
> To: 'Dunaway, Dave'
> Cc: 'Cytometry List'
> Subject: Re: [Cytometry] Problem with BD CS&T laser settings
> 
> Hi, Dave!
> 
> CS&T is the bead based Cytometer Setup & Tracking algorithms BD has
> introduced in the DiVa6 software to automate setting laser delay and
> scaling, and optimal PMT voltages. It is based on the paper by Holden Maeker
> and Joe Trotter "Flow Cytometry Controls, Instrument Setup, and the
> Determination of Positivity" Cytometry Part A 69A:1037-1042 (2006).
> 
> If you haven't seen this system, I assume you are running DIVa version 4 or
> 5 still?
> 
> I firmly believe that users should not be allowed to make delay or config
> changes, which is why this dialogue box can wreck so much havoc.
> Unfortunately, if there is a button available, someone will push it no
> matter how many signs you post to the contrary! It's sometimes like a
> child's Saturday cartoon, Tom and Jerry or something.
> 
> Cheers!
> 
> -cbb
> 
> > -----Original Message-----
> > From: Dunaway, Dave [mailto:Dave.Dunaway at nationwidechildrens.org]
> > Sent: Tuesday, July 21, 2009 11:14 AM
> > To: Adrian Smith; Christopher Bare
> > Cc: Cytometry List
> > Subject: RE: [Cytometry] Problem with BD CS&T laser settings
> >
> > Chris,
> >
> > What is CS&T?  I currently have 3 LSR's in our core running DIVA and I
> > don't even know what you are talking about.  We have self-users as well
> > but nobody but me makes any adjustments to delays or scaling.  Users run
> > beads for Quality Control to assure that the machines are operating
> > properly but nothing more than that.  Why would someone want to go in
> > and change the delay if your QC looks good across all lasers and PMT's.
> > Too many "button pushers" out there to allow the users access to these
> > adjustments let alone anything to do with changing configs.
> >
> > Dave Dunaway
> > Nationwide Childrens Hospital Research Institute
> >
> > -----Original Message-----
> > From: cytometry-bounces at lists.purdue.edu
> > [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Adrian Smith
> > Sent: Tuesday, July 21, 2009 10:54 AM
> > To: Christopher Bare
> > Cc: 'Cytometry List'
> > Subject: Re: [Cytometry] Problem with BD CS&T laser settings
> >
> > Hi Chris,
> >
> > Thanks so much for writing this up in so much detail for everyone.
> >
> > We had come across the same problem recently and have resorted to
> > pasting the correct laser delays on a card on the instrument so that
> > people can double-check they have the right settings before they
> > start. Most of them have been good at choosing to accept CS&T settings.
> >
> > In fact, it seems that this may be an extension of problem we first
> > noticed with the introduction of the user controls in Diva 5.
> > Basically it was possible then to get into the situation where someone
> > could have the laser settings changed under them with no ability to
> > change them back. The situation where that could happen was much more
> > limited (basically you had to have a some users with full or partial
> > permission to change some settings, and some users with no
> > permissions) so maybe it wasn't common.
> >
> > I did submit a detailed report to BD but don't believe I heard back -
> > and there are clearly some additional traps in Diva 6 that are going
> > to catch a lot more people.
> >
> > (I can't remember if I sent a report to list - I thought I might have
> > but I can't find anything in the archives).
> >
> > Regards,
> >
> > Adrian Smith
> > Centenary Institute, Sydney, Australia
> >
> >
> > On 21/07/2009, at 5:18 AM, Christopher Bare wrote:
> >
> > > 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
> > > https://lists.purdue.edu/mailman/listinfo/cytometry
> >
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