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Sudden signal Intensity drop during Helios runs

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cguidos

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Joined: Tue Nov 18, 2014 3:10 am

Post Fri Feb 09, 2018 9:58 pm

Sudden signal Intensity drop during Helios runs

Dear Mike, Greg B and anyone else who is interested.

This is an update to our Signal Drop problem previously mentioned in the "Signal variation upon freezing samples" thread from January. As I mentioned then, we have been experiencing this problem intermittently for some time and it is unrelated to sample preparation. I have attached a PDF showing examples from recent experiments where we ran technical replicates of a PBMC sample on 2 different days.

The cells were stained with Abs, Cisplatin and Iridium (overnight) in 1 batch and then split into 10 tubes. Five were acquired on day 1 on our Helios 1 after a full clean. The other 5 were stored in Ir and acquired on day 2. I have not included all markers - just enough to show that some are affected by a sudden and dramatic signal drop (eg., 89Y, , others are slightly affected while others not all (only a few: 141Pr, 151Eu, 153Eu). Sometimes the drop happens early and sometimes late. Sometimes the signal is clearly split for the entire acquisition (eg. Tube 1 day 2) but that is rarer than the drop partway through.

Finally, sometimes the drop is present for the entire acquisition -ie the signal in the affected channels is abnormally low. We can only know this is a drop (as opposed to poor stain) when we have technical replicates - see the last slide from a run on our 2nd Helios (same cells and Abs). You see that the CD4589Y and CD3-170Er intensities are very low in tubes 1 and 2, but start higher in tube 3 and then drop.

Needless to say this has been very frustrating since it came to light. Not sure when it started but within the last year. We stopped checking Time plots routinely since things had been quite stable. Fluidigm has not identified the problem but thinks it is caused by some sort of (sporadic) cell instability. They asked to test some different acquisition conditions, the details of which I can't divulge. These conditions provided a notable improvement in signal quality (increased tightness of positive peaks) compared to the baseline samples. However, we did not see our particular signal drop problem in the single samples we ran under our conditions.

So the cause remains a mystery. We will now use Fluidigm's new conditions routinely and see if that solves the problem. Will keep you posted!

Cindy

PS - also note the increased background in HLA-DR after 48 h storage in Ir - we have this problem with a few Abs upon storing stained fixed cells - any ideas on how to solve that?
Attachments
Signal Drop Issue SickKids Helios Instruments.pdf
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GregBehbehani

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Posts: 85

Joined: Tue Apr 12, 2016 10:17 pm

Location: The Ohio State University, Columbus, Ohio

Post Fri Feb 09, 2018 11:10 pm

Re: Sudden signal Intensity drop during Helios runs

Hi Cindy,

Wow; so frustrating. I assume, as before, there is no change in the bead signal when this is happening.

Some suggestions:

1. Did you ever test to see if this would ever happen in solution mode?

2. If you have Cytobank (or something else that create pseudo-3D-plots), try coloring the CD45 vs. time and the CD3 vs. time plots from Tube 1 day 2 for all of the other markers that you measured. Do you see any markers that are preferentially present in the cells with the lower signal?

3. Finally, did you ever test the stability of your samples in pure water (i.e. just sitting in water without running in the machine). I still think this phenomenon is most likely due to something that's happening to your cells rather than your CyTOF. From the previous post, I would try the following (if you haven't done it already):
"-Regardless of the cause, this should be fairly easy to sort out, as this effect would be dependent on the time the cells have been diluted in pure water. I would suggest repeating the experiment, but this time dilute both the frozen and never frozen cells in aliquots of pure water and let them sit at room temp for different periods of time (1m, 5m, 10m,....60m, etc.). If some sort of physical degradation is at play, the longer the cells sit diluted in pure water the greater the difference will be between frozen and never frozen samples (irrespective of whether or not they are running in the machine)."

If this is a fixation problem, it will likely seem intermittent, (at least this has been our experience). Lots of things influence the post-staining fixation (time PFA/fixative has been open, temperature, amount of cells and protein, residual volume of staining media when the fixative/Ir is added, how the cells were permeabilized, etc.), the combination of all of them can lead to effects that seem random.

Good luck,

Greg
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cguidos

Contributor

Posts: 28

Joined: Tue Nov 18, 2014 3:10 am

Post Fri Feb 09, 2018 11:17 pm

Re: Sudden signal Intensity drop during Helios runs

Hi Greg

You are right the bead signals do not shift. Interestingly 165Ho is one of our affected channels - you can see the 165Ho marker on cells shift but beads do not.

We certainly have seen all signals degrade with prolonged sitting in pure water but not selectively for certain tags as we saw here. I also don't think it is happening preferentially to some subset of cells with different characteristics - have seen it with many different types of cells including cell lines. Always the same tags degrade, regardless of which markers or cell types were stained.

Thanks for your thoughts -we'll keep plugging away

Cindy
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GregBehbehani

Master

Posts: 85

Joined: Tue Apr 12, 2016 10:17 pm

Location: The Ohio State University, Columbus, Ohio

Post Fri Feb 09, 2018 11:54 pm

Re: Sudden signal Intensity drop during Helios runs

Hi Cindy,

That's really helpful information. I think the problem is your antibodies.

A few experiments you might try to sort this out:

1. I would still try the water incubation experiment, but in this case you'd be looking for your antibodies to fall off, rather than for the cells to fall apart. The fixation during the Ir incubation is primarily to fix the antibodies in place (though it also helps keep the cells together). Some antibodies can hold onto their antigens in pure water, while other's can't. If you have certain antibodies that can't stay attached (via normal protein-protein interactions) in pure water and then they don't get fixed in place well enough, they'll fall off at some random time after being put into pure water. Again, I would fully expect it to seem random, except that it would always get worse during a run (never start worse and get better) and only certain antibody clones would *sometimes* exhibit this behavior.

2. If a water incubation does reproduce the phenomenon, you could then try using IgG capture beads to differentiate between the antibody falling off and the metal falling off the antibody (in the former case the bead-bound antibody will be stable in water, while the in the later, the drop will still happen on the bead).

3. To further sort out antibody vs. cell, you could try testing a different antibody clone against the same antigen (it's likely to be more stable), or an antibody against a different antigen of the same membrane protein (this will stay bound while the water sensitive antibody will fall off).

I'd be very interested to know what you find out.

best,

Greg

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