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Periodic fluctuations in events/sec acquisition

PostPosted: Mon Nov 29, 2021 2:41 pm
by TomerWeizmann
Dear CyTOFers,

We have recently observed a fault of periodic fluctuations in events/sec acquisition (and a delay in on-the-fly acquisition, which may take over 45min for 1E6 events to complete recording after the run has been stopped).

Please see attached (slides 6-8 are the same cells, provided by FDM, aquired in Markham vs on our Helios).

Two FDM FSEs visited our site, diagnosed the fault, but could not resolve it (multiple parts were replaced, including the PC and PSI).

FDM is investigating.

We would appreciate any advice, and if you can please also check your data for these fluctuations.

Sincerely,
Tomer

Re: Periodic fluctuations in events/sec acquisition

PostPosted: Tue Nov 30, 2021 5:25 pm
by desireeBCRT
Hi Tomer,
this looks scary. In combination with the delay in on-the-fly acquisition I would guess software/PC issues, but you said that the PC has been changed already, right?
What about the pressure, were there any changes in the status panel visible during acquisition related to these fluctuations?

Kind regards
Desiree

Re: Periodic fluctuations in events/sec acquisition

PostPosted: Wed Dec 01, 2021 10:55 am
by TomerWeizmann
Thank you Desiree,

Indeed.
Yes – PC replaced.
The pressure was stable and validated via the FSE using inline gas flowmeters.

The mystery remains,
Tomer

Re: Periodic fluctuations in events/sec acquisition

PostPosted: Wed Dec 01, 2021 3:38 pm
by mleipold
Hi Tomer,

It's weird that it's as repetitive as it is. If these were random fluctations, you'd expect random spikes. When we've had a wonky gas supply manifold, it's been jumpy, but not as repetitive as what you're seeing.

The spike appears to be a *spike*, rather than a momentary return to normal. Most often you see this with a partially clogged nebulizer bursting through, though formally any increase in flow rate (PSI argon pressure spike from a misbehaving gas manifold or partially clogged flow sensor), etc) would cause it.

Also, it seems unlikely to be a vacuum pump issue, or a cone o-ring issue: those usually manifest as fairly consistent overall low signal, as does a skimmer needing to be replaced or a broken skimmer charging connection (the copper thing the skimmer screws down against).

A few questions:
1. Even though you've changed your PSI, do you have a Supersampler to test? This would help isolate once and for all that the PSI itself (and its associated gas flow and flow meter) isn't the problem.
- I bring this up because we did have one instance in which a new PSI had to be swapped out *again* in less than a month, due to either an irreversible clog in the flow sensor or the flow sensor just became unreliable.

2. Have you tried changing the Nebulizer gas line?
3. Have you tried changing out the spray chamber (unfortunately, you need to order that part even if you have a service contract)?
- 2 and 3 would help make sure the issue is *inside* the instrument, rather than something the user can touch.

4. Do you see the same spiking with Tuning Solution, or is it just with particulates (Beads, Cells, etc)?


Mike

PS: I will say, your Bead signal on Slide 1 (consistent flow, before 17 Oct 2021) seems low. Was the instrument warmed up and tuned before this measurement was taken? In our hands, Ce140 signal is always above 1e3 if the instrument is tuned properly. In fact, we use that as a metric while running: if the Beads start dropped to 1e3 or below (long run, dirty sample, etc), we stop, Wash, and then re-Tune.....usually at least the DV has changed significantly.

Re: Periodic fluctuations in events/sec acquisition

PostPosted: Wed Dec 01, 2021 5:36 pm
by TomerWeizmann
Thank you Mike,

We were waiting for your feedback :-)

Answers:
1. We only have PSIs. Tested three.
2. Yes. Several times. As well everything else we could think of.
3. No. I agree and suggested that, but did not have the support of FDM service (our system is under warranty). We do not have a spare one on site, but I will ask to get one – it is definitely worth a try. Thanks!
4. Yes, but it is much more prominent with particulates. Please see attached (ts_29Nov2021_0959).
P.S. – Correct, that was taken after changing from HT to WB, pre-tuning. I have attached a typical output (beads1_29Nov2021_0920).

The current hypothesis of FDM is that this results from faulty (in manufacturing) nebulizers and capillaries (though we tested quite a few), which causes the formation of bubbles. They intend to send us validated sets to test.

Take care,
Tomer

Re: Periodic fluctuations in events/sec acquisition

PostPosted: Wed Dec 01, 2021 5:46 pm
by mleipold
Hi Tomer,

Hmmmh. Yes, I can see the fluctuations in the Tuning solution, but that's not anywhere near the magniture of what you see with particulates.


Have you tried removing the grounding nut and sonicating it?

If there's a weird clog inside the grounding nut orifice, then that would allow Solution (Tuning, etc) to pass through relatively unimpeded, but not particulates. We've had that happen once or twice. MilliQ alone would be fine; the Pearl solution (80% 5% citranox, 20% Methanol) would probably also work....the methanol would probably keep the detergent bubbles down. If not, then 50/50 MeOH/water is something that we used regularly to do a deep clean on FPLCs. You'd definitely want some kind of organic or detergent in there to help remove cell residue.


Mike

Re: Periodic fluctuations in events/sec acquisition

PostPosted: Wed Dec 01, 2021 5:52 pm
by TomerWeizmann
That’s a good idea.

We have definitely cleaned it well by flushing, but did not sonicate it. We have Citranox and MeOH. Will try and update.

Many thanks,
Tomer

Re: Periodic fluctuations in events/sec acquisition

PostPosted: Wed Dec 01, 2021 5:59 pm
by dahern
We recently had a very similar readout as your tuning screenshot above - very repetitive peaks and troughs.

If we looked at the spray from the nebuliser very closely we could see a very subtle repetitive change in the spray that matched the timings of the tuning readout. Having a look at that closely might help you see if the problem is before or after the nebuliser.

We changed much of the same things as you have and saw it over multiple capillary lines and nebulisers, though to various degrees. Ours either disappeared by itself (and our swapping of the nebuliser and all the sample lines) or it disappeared when we removed the grounding nut and sonicated it. Might be worth a try.

Re: Periodic fluctuations in events/sec acquisition

PostPosted: Wed Dec 01, 2021 6:04 pm
by eganio
Hi Tomer,

You ight also want to try replacing the nebulizer gas line, as they can sometime become worn out and leaky. We had to do this once when we were having issues getting any signal at all, and tried everything else under the sun we could think of. If you buy the nebulizers directly from Fluidigm, they should come with a new NEB gas line included.

-Ed

Re: Periodic fluctuations in events/sec acquisition

PostPosted: Wed Dec 01, 2021 7:40 pm
by Chowduck
I’ve seen regular pulsing like this from air leaking into the sample arm of the nebulizer. Here’s an extreme example: https://photos.app.goo.gl/SE1bvuAx1Y53PN9w9

David Ahern’s suggestion to watch the neb spray would be informative. Alternatively, if you can get ahold of a pocket microscope you might be able to see bubbles in the neb capillary.

Greg Chang