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CyTOF quality data check

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Posts: 4

Joined: Tue Sep 19, 2017 7:52 pm

Post Thu Sep 21, 2017 8:14 pm

CyTOF quality data check


Is it possible to check the quality of your data before clustering/analysis? Is this possible to do if your data is not barcoded?
If so, what program would you use?

(Quality of data in relation to false positives, etc.)

Thanks for your help!




Posts: 2162

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon Sep 25, 2017 8:57 pm

Re: CyTOF quality data check

Hi Michelle,

I know that you and I have talked about this in email last week (around the timestamp of this post that was delayed), but as a public answer on Cytoforum: It really depends on what you mean by quality check.

If you are asking about something like a Levey-Jennings plot in flow, you would need something like a plot of daily tuning values over days/weeks. Unless you have your own machine(s), you're unlikely to have this information (though the CyTOF manager of your instrument could probably get it to you).

The only stuff you would have in your actual sample FCS files would be the EQ 4-element normalization beads: you could track that information in your samples. Ce140, Ce142, Eu151, Eu153, Ho165, Lu175, and Lu176 are all present in the beads. However, that information has some limitations as well: all the signals except for the Ce142 and Lu176 are really bright, so it wouldn't be that informative on the sensitivity at the lower signal range. The beads are also a "fixed" reagent that is really hard to screw up; you'd have to soak the beads in nitric acid or something to start changing their signal.

This is in contrast to your sample reagents: you could have a pipetting error, a low-quality sample that affects "effective" reagent titer, a weaker prep of antibody than usual, poor washing efficiency, etc. All of these affect both "true" signal intensity as well as the background of each channel in your sample. To address at least some of these issues, I validate (and if necessary, retiter) all in-house conjugates. I also run a healthy standard control sample on every plate to make sure I didn't screw something up (basically, it's a processing control). I make sure that *its* signal intensity and Freq Parent values are consistent with previous results; if so, than any variation seen in the non-control samples should be "true" (including differences due to biology, disease state, initial processing like Ficoll and storage, etc).


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