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Varying intensity in some channels

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juliam

Participant

Posts: 18

Joined: Wed Oct 07, 2020 8:13 am

Post Tue Oct 13, 2020 9:30 pm

Varying intensity in some channels

Hello Everyone,
I have problem with data interpretation.
In my experiemnt I had control mice and treated mice.
I noticed that intensity of some channels (channel 166Er used for EpCam) for 2 of control mice was way stronger (more than 10 folds) when compared to rest of samples.
Surprisingly also another marker (functional marker) in channel 169Tm is very strong in these 2 samples.
Based on intensity of marker in 169Tm I am quantifying one of most important readout. However I am wondering whether my readout in 169Tm is simply affected by change in intensity of 166Er?? How to normalize it?
I normalized data with Fluidigm software.
I would be extremely happy for suggestions, and help.
best wishes,
julia
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Tue Oct 13, 2020 9:45 pm

Re: Varying intensity in some channels

Hi Julia,

Before you think about doing some kind of batch correction on your samples, you first have to establish that it truly is false signal.

A couple comments and questions:

1. 166Er and 169Tm are different elements. Erbium and Thulium share no isotopes in common, therefore they cannot give isotopic spillover to each other. And 166 is far enough away from 169 that you're not going to have Mass Resolution-related right or left "leg" spillover, no matter how high the signal is in either channel.
-therefore, I don't see any way that you can have spillover due to the metals.

2. When you were acquiring the samples, did you see background signal "streaking" in the 166 or 169 channels, that might result in higher background? If you did *not* see heavy streaking (ie, the only signal seen was associated with punctate, resolved cell events), then that can't explain it.

3. Therefore, one explanation is that this difference is genuine biology for these 2 control mice. What reason do you have to exclude that possibility? Were these two control mice stained in a separate experiment from all the others, or something?


Mike
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antonio

Contributor

Posts: 20

Joined: Wed Dec 04, 2013 3:05 pm

Post Thu Oct 15, 2020 4:31 pm

Re: Varying intensity in some channels

Dear Julia,

maybe you can share a plot of 166 versus 169 patterns?

Best
Antonio
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juliam

Participant

Posts: 18

Joined: Wed Oct 07, 2020 8:13 am

Post Tue Nov 17, 2020 2:54 pm

Re: Varying intensity in some channels

Hi Mike, Hi Antonio,

Thanks for your reply. I really didnt know how to comment as in theory there should not be spillover, but there is somewhing bizarre.

Recently Idid run different panel but also consisting of CD31 165Ho, CD326 166Er, p16 169Tm, and I do face similar problem with one sample. Signal for Cd31 is very high for one sample which shows surprisingly high signal in 169Tm channel. Its simply suspicious as always this surprisinly high signal in 169Tm is assisted with shift in signal intensity of 165Ho.
All samples were prepared together and antibody mix as well.

For some reason I am facing problems to attach graph, therefore I would be happy to email them if You Mike or Antonio would like to take a look.

best regards,

Julia
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Tue Nov 17, 2020 4:09 pm

Re: Varying intensity in some channels

Hi Julia,

There's a size limit to the attachments (a few hundred KB, but less than 1GB). So, that may part of your attachment problem.

But you can always post it to Google Drive or something and post a link to it.


Mike
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juliam

Participant

Posts: 18

Joined: Wed Oct 07, 2020 8:13 am

Post Wed Nov 18, 2020 11:50 am

Re: Varying intensity in some channels

Hi MIke,

this is the link to google drive files

https://drive.google.com/drive/folders/ ... sp=sharing
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed Nov 18, 2020 4:36 pm

Re: Varying intensity in some channels

Hi Julia,

OK, from that Google Drive link and your comment in an offline email, I put this PDF together.


Honestly, if you prepared enough cocktail for the entire sample batch, and Sample 1 is the only shifted one, then I have to assume has something to do with that one sample.

This could be biology, sample processing/storage, contamination specific to that sample (metal, etc), or some combination of them all.

Particularly in the lower left (Sample 1, 165 vs 169), your boxed population looks like it's a discrete population with fairly constant CD31 expression but higher p16 expression. If it were some kind of spillover, then it would likely be more on a diagonal.


Mike
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