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CyTOF Data Issues

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fantabarrow

Participant

Posts: 4

Joined: Wed Jul 22, 2020 9:32 pm

Post Wed Jul 22, 2020 10:56 pm

CyTOF Data Issues

Hello all,

I have been using CyTOF2 for a year now with a 26 metal panel and I recently bumped that number up to 29 metals. However, after acquiring the data and normalization, the fcs files looked weird. I am hoping someone could please shed some light into what the issue is and how it can be fixed. I have attached a snapshot of one of the fcs files. Thank you.
Attachments
Weird Fcs.JPG
Weird Fcs.JPG (37.33 KiB) Viewed 4360 times
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed Jul 22, 2020 11:29 pm

Re: CyTOF Data Issues

Hi Fanta,

A few questions:

1. Are these Ungated cells, or LiveIntactSinglets?
2. Is the 29 marker panel the same 26 marker panel, plus 3 new markers?
3. What does this file look like before normalization?
4. What does the 26 marker panel look like?


Mike
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fantabarrow

Participant

Posts: 4

Joined: Wed Jul 22, 2020 9:32 pm

Post Wed Jul 22, 2020 11:45 pm

Re: CyTOF Data Issues

Hi Mike,

1. These are live intact singlets
2. The 29 marker panel is the same as the 26 marker panel + 3 new markers
3. I haven't looked into what this file looks like before normalization. I do not have access to the non-normalized sample file right now, I can take a look tomorrow and let you know.
4.
Attachments
26 marker panel.JPG
26 marker panel
26 marker panel.JPG (29.51 KiB) Viewed 4348 times
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed Jul 22, 2020 11:47 pm

Re: CyTOF Data Issues

Hi Fanta,

Thanks for the new picture.

Could you post the panels (Excel, etc)? We have no idea what your spills might be, especially with the addition of the new markers.


Mike
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fantabarrow

Participant

Posts: 4

Joined: Wed Jul 22, 2020 9:32 pm

Post Wed Jul 22, 2020 11:56 pm

Re: CyTOF Data Issues

Hi Mike,

See attached the panels.

Fanta.
Attachments
CyTOF Panel Current.xlsx
(73.24 KiB) Downloaded 264 times
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Jul 23, 2020 4:41 pm

Re: CyTOF Data Issues

Hi Fanta,

Thanks for the panels.

I would strongly recommend that you put the metals in numerical order when you are looking for spillovers. Spillovers are primarily due to elemental impurities of the same mass, isotopic impurities of the same element, and oxide spills. Having them in order makes it a lot easier to see if you have the same element, calculate M+16, and other things.

I have rearranged your table in order, where the green are the 3 new markers and the pink are the two channels in your bivariate plots.

I am assuming that you watched the sample being acquired, so that you can rule out the possibility that the increased background is caused by streakiness due to debris or other sample running or sample quality issues.

Assuming that's true, looking at your panel and your bivariate plots, my guess is that you're getting isotopic impurity spill from your new TIM4-154Sm marker back into the CD3e-152Sm channel. They're the same element, and the next nearest mass, so they would be the most likely to spill.

The simplest way to check this is doing a MMO experiment, where you leave out the TIM4-154Sm but keep all the other 28 antibodies.


Mike
Attachments
CyTOF Panel Current-MDLmod.xlsx
(139.49 KiB) Downloaded 227 times
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fantabarrow

Participant

Posts: 4

Joined: Wed Jul 22, 2020 9:32 pm

Post Thu Jul 23, 2020 5:22 pm

Re: CyTOF Data Issues

Hi Mike,

Thank you for your help with this issue. I will try out the recommendation and let you know how things go. Thank you once again.

Fanta.

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