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CyTOF .fcs files and proper gating

PostPosted: Fri Dec 13, 2013 8:30 pm
by robs
I am looking for some input on any precautions people take when gating CyTOF generated .fcs as opposed to regular flow files.
After speaking with DVS (conversation below) I know that the files are originally generated with only positive integer values and zero but then it is randomized by the software.
I ran an .fcs file back through the software and told it to restore the originally acquired values (plot on the left) and I compared it with the automatically randomized data (plot on right). If I gate between zero and one I get different population values, obviously because of the distribution

original versus randomized.png
original cytof generated file versus uniform negaive randomized data


Would just never gating between zero and one prevent this problem? If the same file is randomized on different occasions using the same algorithim is it possible we would get significantly different gate stats, e.g. % parent, median, between the different randomizations? If so, would using rectangular gates, instead of freeform, prevent this?

Thanks for any thoughts you guys might have,
Rob



P.S. Here are some answers I got from the DVS people about .fcs file generation. Their answers are italicized

1. Has DVS released any technical note regarding how the push data is converted to FCS.
No tech note yet (but that is a really good idea). Here is a brief description. It is the two stage process: firstly, cellular events are separated from the background. It is done using a simple thresholding reflected in the Acquisition window/Analysis tab as the Lower convolution threshold for the total (sum of all channels) ion signal. This stage is key in localizing the cellular events in time assigning "start push-end push" boundaries to every event. On the second stage, the ion signal is integrated from the "start push" to the "end push" over all channels separately keeping "end pushes" minus "start push" value as an event length. These data are converted to FCS.


2. The original CyTOF data would be all integers of ion counts per cell, right?
Correct.

3. If that’s the case what kind of randomization is used to get the non-integer values that I see in the .fcs?
The default randomization during the direct acquisition is the same as in the FCS Analysis window: the randomization is applied to all values using the uniform negative distribution. That means that the ion count value 1 is uniformly distributed in the interval ]0,1] ( funny brackets mean "excluding zero and including one"). The same for any other the ion count value: for example, 10 counts will be uniformly distributed in the interval ]9,10].


4. The software allows us to generate .fcs files with custom data randomization settings after the fact, but I could not find what the default values are that come with the on-the-fly analysis in the acquisition window.
We will get back to you on this, we should make it clear in the SW Manual.


5. Does the conversion to .fcs bin the data into a certain number of channels in a particular range, like Diva does? or are the 'true' ion counts reflected in the CyTOF .fcs file?
We do not bin data artificially at all. The randomization can be switched off in post-processing of the FCS file and the raw data will be available with all complications of its presentation. Notice, that in our Plotviewer program we employ the unequal binning procedure which delegates different screen "real estate" to different ion values. Therefore, only one bin is dedicated to ]0,1] interval as well as for the ]10,11] interval (for example). In this case, the randomization is not required and the problem does not exist.

Re: CyTOF .fcs files and proper gating

PostPosted: Fri Dec 13, 2013 8:43 pm
by mleipold
Interesting question.

What would happen to the left plot if you also grabbed the "streak" of events immediately to the right of your gate (Dual=11?), into your gate? Does that give you the extra ~8%?

Re: CyTOF .fcs files and proper gating

PostPosted: Fri Dec 13, 2013 9:43 pm
by robs
Thanks for taking a look.
Yes.

shifted gate.png


The numbers are still slightly higher for the randomized because I can't set gate coordinates exactly to 1 in FlowJo7. As a consequence, some of the randomized events between 1-2 are sneaking into the gate in the randomized file.

Not sure how important this all is to keep in mind.

Re: CyTOF .fcs files and proper gating

PostPosted: Fri Dec 13, 2013 11:38 pm
by mleipold
I don't think this is that important, in the regular usage of the FCS files.

I can't think of a time when I've ever gated the 0-1 population as separate from, say, the 1-10 population.

Part of it is that the typical "background" (rather than "true") signal for a marker is usually anything below 5 (usually 10) Dd counts (CyTOFv1). Therefore, my "negative" gates always encompass the events in the (-1<0<1) range.

Re: CyTOF .fcs files and proper gating

PostPosted: Sat Dec 14, 2013 6:51 am
by Ofir
Very good point Rob. The data randomization process was implemented in order to allow data analysis with visual software used for Flow Cytometry (FC). This is important to allow FC to users naturally transition of Mass Cytometry (MC).
As you know, MC is more than just FC enhanced - the real value is in analyzing MC data in multidimensional space rather than extensive gating on two parameters at a time.
Therefore, if you are using any of the multivariate analysis tools (e.g., SPADE and viSNE) you should use the original (non-transformed) data files as your input.

It is my personal opinion that when presenting dot plots the transformed data should be used. It is easier to see populations - as defined by a central value with distribution - and is the historical standard for display.

Re: CyTOF .fcs files and proper gating

PostPosted: Mon Dec 16, 2013 3:23 pm
by robs
Thank you guys for your insights, our CyTOF team is expanding so I want to be able to intelligently explain these differences in display to the newcomers.

Re: CyTOF .fcs files and proper gating

PostPosted: Mon Dec 16, 2013 4:14 pm
by mleipold
Ofir,

In order to get the non-transformed data, you'd have to take the CyTOF output and back convert it.

I don't know of anyone who does this. Everyone I know takes the output FCS files and after gating out the debris/doublets/dead cells, re-outputs FCS files from FlowJo or whatever, and then uses those as the input for SPADE.

Re: CyTOF .fcs files and proper gating

PostPosted: Mon Dec 16, 2013 7:58 pm
by Ofir
Hi Mike, I have heard different. I wonder if you could get someone from the Nolan lab to chime in on how the original SPADE paper was done.
It kind of makes sense to me to use the original data without the added "noise". I'll try running SPADE both ways and report back.

Re: CyTOF .fcs files and proper gating

PostPosted: Thu Jan 23, 2014 4:02 am
by ndawson
*bump* Did you find anything in your SPADE analysis, Ofir?