[Cytometry] Problem with BD CS&T laser settings

Melissa Makris mmakris at vt.edu
Fri Jul 24 12:04:23 EDT 2009


Hi All,

I'm really enjoying reading this thread.  

I have to side with those that are saying to leave the voltages set by CS&T
in place.  Unless, of course, the positive population is off scale.  Then
you would need to turn down the voltage just enough to bring those positives
back on scale.  

Using cellular autofluorescence to determine PMT settings isn't as accurate
as we might like.  With the newer digital instruments, it is better to
optimize each channel of interest by focusing on the positive population.  

CS&T does this - which also makes sure that the unstained population is
above the electronic noise so dim populations can be adequately resolved
from background.   

(Before CS&T came out, BD had a whitepaper on how to optimize voltages
manually on their digital instruments.  It was quite a tedious chore, so
thank goodness they automated it!)

Robert makes some excellent points.  The technology has changed, and we need
to change with it.  We need to get used to seeing unstained cells higher up
on the axis.  Just like we have to get used to seeing all those cells under
the axis.  It's all in the name of better data. 

Best regards,

Melissa

-----------------------------------------------
Melissa R. Makris
Flow Cytometry Lab Supervisor
Virginia Tech
College of Veterinary Medicine
Duckpond Drive, Phase II 0442
Blacksburg, VA 24061
(540) 231-4115
-----------------------------------------------


-----Original Message-----
From: cytometry-bounces at lists.purdue.edu
[mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Robert Wadley
Sent: Thursday, July 23, 2009 7:01 PM
To: chris.willberg at ndm.ox.ac.uk; cytometry at lists.purdue.edu
Subject: Re: [Cytometry] Problem with BD CS&T laser settings


Hi All,

I have had advice similar to Chris.  The voltages set by the CST are
apparently less to do with the beads but with the beginning of the linear
response of the relevant PMT.  My understanding is that while you can reset
the baseline voltage based on the autofluorescence of your cells you should
be mindful that by lowering the voltage your PMT signal for the
autofluorescence is no longer in the linear "portion" of the relevant PMT.  

I guess in most cases this would not be particularly relevant, especially
with bright fluorochromes.  Alternatively by raising the voltage you reduce
the linear "portion" of the PMT that you can play with.

 

In the 'digital' age do we really need to have all our autofluorescent MFIs
to be similar?  Most of us put our autofluorescent peak in a particular
position because that is what we did on analog systems.  Doesn't digital
technology allow us more flexibility?  Is it more important to work within
the linear 'portion' of our PMTs than set our autofluorescent peak in a
particular position?

 

I have used CST on my Aria and love it, until a loose sheath line showered
PBS over the fluidics board!  Hopefully my Aria 1 to Aria 2 upgrade will let
CST work again.

I am trying to get CST working on my LSR II, but have run into a few
problems with Administrator Permissions that I have to work out with my IT
department.

In general my view is that it is my job to run CST, and the users don't play
with it.  The exception to this rule is likely to be the Clinical Trials
Team, who have specific QC requirements and will most likely end up runing
CST prior to their experiments.

 

Regards

 

Robert Wadley

Cytometry Manager

Cytometry & Imaging Suite

Mater Medical Research Institute

Brisbane, Australia
 
> From: Chris.Willberg at ndm.ox.ac.uk
> To: cytometry at lists.purdue.edu
> Date: Thu, 23 Jul 2009 10:33:33 +0100
> Subject: Re: [Cytometry] Problem with BD CS&T laser settings
> 
> 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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