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Decrease in signal and tuning stability

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Verhoeff

Participant

Posts: 16

Joined: Wed Mar 02, 2016 10:25 am

Post Mon Dec 23, 2019 1:05 pm

Decrease in signal and tuning stability

Hi all,

Lately we've been experiencing unexplained signal drops during runs that seem unrelated to antibody or sample integrity. Samples were stained, fixed and run consecutively, but then some acquisition runs see a sharp dropoff in signal after about 10-15minutes. First time it happened we had 1 day of problem-free runs, then the second day saw signal drop offs:
Tuning instability.PNG
In between these consecutive runs we performed Full tuning, where QC passed with slight adjustments to XY and/or neb+makeup gases.


PSI pressure stayed constant, giving no indication of clogs or faulty nebulizer spray. Every sample was washed with H2O just prior to running, the same as the day before where no such issues occurred. When during the final run signal actually rebounded we stopped acquisition and called it quits for the day.

Week after with a different user we saw a similar pattern, after startup and Tuning (with QC values well within spec) sample values noticably dropped after a short time.
Signal-drop.PNG
Note the sudden increase in CV in 175Lu bead signal and 198Pt live/dead stain

First sample was fine, second sample experiences a significant drop in antibody signal and interestingly a widened platinum and EQbead peak. Running full tuning right after again results in a passing QC.

All our users use fresh PFA fixations after antibody staining steps, so release of metal from the cells is unlikely.

Have any other labs seen issues like these where Tuning would pass but proves relatively unstable?

Thanks,
Jan
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eganio

Participant

Posts: 15

Joined: Wed Nov 07, 2018 8:00 pm

Post Fri Dec 27, 2019 5:57 pm

Re: Decrease in signal and tuning stability

Hi Jan,

Can you also post your tuning parameters? Like you said, this seems like an instrument issue, rather than a staining or sample integrity issue. Knowing how the instrument is tuning might help diagnose what's going on. If you can take a screen shot of the tuning results from the last time you had the issue, that would be helpful.

-Ed
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jimbomahoney

Master

Posts: 73

Joined: Wed Feb 27, 2019 11:21 am

Post Mon Jan 06, 2020 12:39 pm

Re: Decrease in signal and tuning stability

Hi Jan,

It might also be useful to know which instrument version and which injector you're using?

It might also be worth taking note of the vacuum pressures, although ideally you'd be able to compare the pressures during a good run vs. a bad. (This is a stab in the dark on my part, based on another thread).

James

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