[Cytometry] Problem with BD CS&T laser settings

Chris Willberg Chris.Willberg at ndm.ox.ac.uk
Thu Jul 23 05:33:33 EDT 2009

Hi All,

Sorry this may be a very basic question. I was told not to change the  
PMT voltages (except the fsc and ssc) after the CS&T had set them -  
unless your positive stain was off scale. This results in some  
negative populations being quiet "high" up on the scale. However, I  
was warned that if you lower the voltages you will lower the  
sensitivity to discriminate between negative and dim populations.  
Until now I have been happy doing this. Is this correct? What is the  
best practice?

Many thanks
Chris Willberg

Chris Willberg
Postdoctoral Research Assistant
Biomedical Research Centre - Immunology Theme
University of Oxford
JR Level 7
Room 7602
Oxford, UK

Tele: 01865 271290
or tele: 01865 221341/221339
e-mail: Chris.Willberg at ndm.ox.ac.uk

On 22 Jul 2009, at 22:32, Uriel TK wrote:

> 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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