[Cytometry] Problem with BD CS&T laser settings

Sasha Lazetic Sasha.Lazetic at oncomed.com
Thu Jul 23 18:16:19 EDT 2009


I'll second Ian's statement and proceed a little further.

As was mentioned by Uriel, Felix Heymann and Chris Bare in this thread
use the CST for delay/scaling and set your PMT voltages yourself:

Felix:
> 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.\

Chris:
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.

Uriel:
> 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.

Cell size, aggregation, auto-fluorescence, FcRgamma expression
variability can all vary greatly from population to population, and this
will impact the placement of your negative control and resulting
compensation. Generally I'll set my PMT voltages on digital instruments
to place the unstained/negative control population somewhere between
25-100 MFI (5 or so for analog instruments) for all colors relevant in
your experiment after using the CST settings for laser delay and area
scaling. I try to keep the negative control at a consistent MFI from
experiment to experiment for the same population of cells.

I'll be blunt and say whoever told you changing the PMT voltages
decreases your sensitivity is incorrect. Changing PMT's does not change
your instrument sensitivity relative to the spread between your negative
and positive samples. Increasing the voltages just increases the signal
(MFI) of both your negative and positive samples. However, increasing
the voltage for a particular or all PMTs forces the user to increase the
compensation for that color(s). You can test this using Calibrite beads.
Combine unlabeled beads with FITC or PE etc labeled beads and set
voltages/comp and then increase the voltage for the PMT detecting your
labeled bead and see what happens.

I prefer having the users of our facility understand PMT voltage and
compensation settings. Hopefully this helps and I'm (mostly) correct.
Howard, Mario, and a multitude of others on this list can elaborate
and/or correct me if I'm wrong.

Cheers,
Sasha Lazetic
OncoMed Pharmaceuticals


-----Original Message-----
From: cytometry-bounces at lists.purdue.edu
[mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of Ian Dimmick
Sent: Thursday, July 23, 2009 12:41 PM
To: Chris Willberg; Cytometry List
Subject: Re: [Cytometry] Problem with BD CS&T laser settings

This is a blanket statement that will not be applicable to all lasers,
or all cells , I think I would go as far as to say it is incorrect 
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 Chris Willberg
[Chris.Willberg at ndm.ox.ac.uk]
Sent: 23 July 2009 10:33
To: Cytometry List
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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