[Cytometry] Problem with BD CS&T laser settings

Felix Heymann felix.reichel at ukb.uni-bonn.de
Tue Jul 21 12:35:26 EDT 2009


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
>
>
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> Cytometry at lists.purdue.edu
> https://lists.purdue.edu/mailman/listinfo/cytometry
>
>   



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