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Where did my cells go???

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Ofir

Master

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Joined: Thu Nov 07, 2013 12:46 pm

Location: US, CA

Post Wed Nov 27, 2013 9:11 pm

Where did my cells go???

By now it's a well known fact that in CyTOF much of the sample is lost on its way to the detector. This can be as much as 50% (although typically less).
Cell loss happens mostly in the stage where cells are desiccated in the heater and injected into the plasma through the two cones called skimmer-reducer. But knowing that is not much comfort.

There are two workarounds for this issue. One is to inject a larger sample using the syringe pump (or other sample introduction method if you are feeling bold). Yet another is running several "repeats" of the sample and stitching together the resulting FCS files. Although I've never tried it myself the Broad Institute's GenePattern is supposed to have this feature.

What's your trick for getting more cell reads?
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mleipold

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Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed Nov 27, 2013 9:22 pm

Re: Where did my cells go???

Hi Ofir,

There is a distinction between CELL transmission efficiency, and ION transmission efficiency.

CELL transmission efficiency is primarily in the fluidics in the front of the machine, BEFORE the plasma. So, cells sticking to the tubing, nebulizer, and spray chamber. Clogged tubing will reduce your number of cell events, but the *signal associated with* those cells will still be accurate. If your machine is clean, tuned, and spraying well, your cell transmission efficiency should be in the 20-30% range.

ION transmission efficiency happens AFTER the plasma. This involves the XY alignment, cones (skimmer, reducer), and ion optics such as the quadrupole mass filter. Typically, for a properly tuned and adjusted machine (CyTOFv1), this is 1/10,000 (put in 10,000 ions, and detect 1).


Because of the cell transmission efficiency issue, you need to run larger samples to find rare cell populations (plasmablasts, cytokine-producing cells, antigen-specific cells, etc) than you would if a CyTOF had the efficiency of, say, an LSRII.


Mike
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shenorr

Participant

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Joined: Tue Nov 12, 2013 12:35 pm

Post Thu Nov 28, 2013 3:27 am

Re: Where did my cells go???

Ofir,
There is a utility for merging FCS files on the Nolan lab Github site https://github.com/nolanlab/cytofCore
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robs

Contributor

Posts: 40

Joined: Mon Dec 02, 2013 3:42 pm

Location: University of Connecticut

Post Tue Dec 03, 2013 6:57 pm

Re: Where did my cells go???

Also, Cytobank just came out with a very easy to use free concatenation tool: http://support.cytobank.org/help/kb/cyt ... -fcs-files
Unlike FlowJo, we saw no differences in scaling or display between original and concatenated files when we used the Cytobank utility.
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ianfrank

Contributor

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Joined: Thu Nov 21, 2013 11:59 pm

Location: Tampa, FL

Post Tue Dec 03, 2013 11:34 pm

Re: Where did my cells go???

Flowjo also has the ability to concatenate FCS files.
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Ofir

Master

Posts: 75

Joined: Thu Nov 07, 2013 12:46 pm

Location: US, CA

Post Fri Dec 06, 2013 9:41 pm

Re: Where did my cells go???

Thanks guys!
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mleipold

Guru

Posts: 2156

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri Dec 06, 2013 10:23 pm

Re: Where did my cells go???

Robs,

You said "Unlike FlowJo, we saw no differences in scaling or display between original and concatenated files when we used the Cytobank utility."

What have you seen when FlowJo does the concatenation? On the rare instances when we've done concatenation, we've used the FlowJo utility and haven't noticed any differences, except in the Time dimension.


-Mike
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robs

Contributor

Posts: 40

Joined: Mon Dec 02, 2013 3:42 pm

Location: University of Connecticut

Post Fri Jan 24, 2014 9:49 pm

Re: Where did my cells go???

Sorry I didn't see this until now, Mike.
With some CyTOF generated .fcs when I compare the original data plots to plots from FlowJo concatenated files, some parameters will appear as if they are linearly displayed, even with log scaling (the populations appear very close to zero). Linear parameters like event length and time also appeared truncated and we saw more of the "picket fence" effect, with the same scaling properties. Also, some FlowJo concatenated files looked completely wrong in Cytobank.
These problems were intermittent and how bad they were depended on the version of FlowJo so I decided to just use other tools to keep reproducibility.
-Rob
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mleipold

Guru

Posts: 2156

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri Jan 24, 2014 10:17 pm

Re: Where did my cells go???

Hi Rob,

I've never seen any problems like you're describing. The only dimension I've ever seen affected was Time.

Out of curiosity, which version of the machine, and which software version, were you using when you were seeing this?


Mike
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Ofir

Master

Posts: 75

Joined: Thu Nov 07, 2013 12:46 pm

Location: US, CA

Post Sat Jan 25, 2014 5:24 am

Re: Where did my cells go???

Rob, could you upload a .fcs file so we can have a look?
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