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"New" cones from fluidigm

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markyyy

Participant

Posts: 15

Joined: Wed Jul 26, 2017 4:09 pm

Post Thu Sep 05, 2019 3:12 pm

"New" cones from fluidigm

Hi all,

We just received a new set of sampler and skimmer/reducer cones purchased from fluidigm. The sampler cone looks brand new; shiny, no abrasions or signs of heat/particulate exposure. The skimmer/reducer on the other hand looks used - and heavily so. I contacted fluidigm to inquire about the wear and they maintain that it is a brand new cone that was "validated" to be within spec for CyTOF2 instruments. Apparently the part is the same for both the helios and CyTOF2, but the CyTOF2 requires testing to ensure CV values are within spec as the it is more sensitive to manufacturing quality of the cones than the Helios.

This raises a couple of questions. First (and most obviously), why is this cone in such poor shape. I am all good with testing to ensure maximal performance and I understand that there might be some slight discoloration from the testing, but I am seeing marks from many repeated installation and removals. In fact, our two year old skimmer/reducer looks significantly less beat up than the one we received. Second, if they are testing cones to be within spec for the CyTOF2, shouldn't the Helios cones also show signs of wear (being that they are essentially the reject cones not up to spec for use on a CyTOF2)? Can any Helios people comment on how their recently purchased skimmer/reducer cones look? third, and this is a more general question that perhaps someone with a better understanding of the way that current is applied across the cones can answer, does engraving an identifier on the face of the skimmer have any effect on its performance? The importance of being gentle when handling/cleaning the cones has been drilled into me since day 1. I understand that this is primarily important around the orifices, but am just curious about peoples thoughts on this.

I plan to test the new cones today and will report back with the results. Would love to hear form others in the meantime about their recent experiences with replacing cones. Thanks so much!

Cheers,
Mark
Attachments
IMG_2977j.jpeg
IMG_2976j.jpeg
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GregBehbehani

Master

Posts: 85

Joined: Tue Apr 12, 2016 10:17 pm

Location: The Ohio State University, Columbus, Ohio

Post Thu Sep 05, 2019 4:28 pm

Re: "New" cones from fluidigm

Hi Mark,

I've never heard of this, but I've also never had a CyTOF2. When I have gotten skimmer/reducers from Fluidigm for my Helios they have looked brand new and we've never had any performance problems with them.

As of the current, it's applied to the reducer (the inner cone) the brown plastic pieces that are around the screws in the back are there to isolate the voltage applied to the reduce from getting to the skimmer. We have intentionally scratched the edge of the skimmer (near the drill holes) on a few occasions without any adverse effects, but we've never done anything as deep as a true engraving would be. We are very particular about how we clean and validate our cones, so if small scratches mattered, I'm sure we would have noticed. I suspect even engraving would be fine, but I think you would have to test it to be sure.

best of luck,

Greg
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eganio

Contributor

Posts: 21

Joined: Wed Nov 07, 2018 8:00 pm

Post Thu Sep 05, 2019 5:22 pm

Re: "New" cones from fluidigm

Hi Mark,

We are using a Helios, and just received a new skimmer/reducer cone. It looks brand new (photos attached). I've ordered several of these, and they've never looked like the one you received. The engraving on the skimmer (exterior) cone seems to be normal, since it is present on ours as well. FYI, I always order the non-tested cone (catalog # 101802). Did you order the tested version (catalog # 107132)? If so, that would explain why it has been used, but I'm not sure why it's so discolored and scratched up. I can't imagine them "testing" it that much!

-Ed
Attachments
New Skimmer Reducer Side.jpg
New Skimmer Reducer Top.jpg
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markyyy

Participant

Posts: 15

Joined: Wed Jul 26, 2017 4:09 pm

Post Thu Sep 12, 2019 6:27 pm

Re: "New" cones from fluidigm

Hi all,

Sorry for the delay in updating on this topic - our instrument has been busy and I was not able to run the tests needed to verify the condition of the new cone sent to us. To address Ed's comment, and as per Fluidigm's recommendation, we did in fact order the "CyTOF 1 and 2 only" cone (cat# 107132). I still cannot understand why testing the cone prior to delivery would result in so much wear and tear. As far as I can tell, the new cone actually performs worse than the new one sent to us. I ran a tuning and beads qc on each back-to-back and found that our old cone was actually outperforming the new one sent to us. I will note - before someone else jumps in to say it for me - that our old cone is indeed still well within spec and that I was not purchasing a new one as a replacement but rather as a backup to have when the time comes.

I am attaching some screenshots from both the tuning and beads-qc for but for visibility-sake here are the numbers that I believe I should be paying attention to when changing cones - please do let me know if there are others that I am not paying attention to. note: I target an oxide ration of 2% as recommended by some members of this forum but otherwise the recommended tuning parameters are applied:

Old cone:
Optimal current = 7.0
Tb mean duals = 935K
RSD duals % = 0.55
CV Eu151 = 11.4
CV Eu153 = 11.8

New cone:
Optimal current = 6.5
Tb mean duals = 901K
RSD duals % = 0.48
CV Eu151 = 13.3
CV Eu153 = 13.8

Based on the above I do not see any significant evidence that the "new" cone is in any better condition than the on we are currently using. I acknowledge that our instrument seems to be running very well with the old cone, but shouldn't I at least see some significant difference in current or Tb value with the new cone? CV values definitely should not be worse, right? By the way, the old cone we are using has been in use for approximately 2 years of what I would call "moderate" usage (weekly usage of 10 to 20 hours per week). Fluidigm has offered to take this new cone back and send us a replacement. The part is currently out of stock which doesn't concern me too much given the quality of our current cone. Any suggestions?

Thanks again for the help in understanding what's goin on here.
Attachments
Newskimmerreducer_tune.png
New cone tuning results
oldskimmerreducer_tune.png
Old cone tuning results
Screen Shot 2019-09-12 at 11.01.04 AM.png
CV values for each cone
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kunicki

Contributor

Posts: 38

Joined: Thu Apr 13, 2017 8:46 pm

Post Thu Sep 12, 2019 11:50 pm

Re: "New" cones from fluidigm

Hi Mark,

I just got a new cone as well, and haven't seen any issues specific to the cone. Maybe this was a one-off issue related to Fluidigm's relocation? The new cone I got did look brand new, and upon installing, the current was at 4 and slowly climbed to 6, depending on tuning.

While trying to diagnose whats going on from the tuning settings between old and new cones, I wouldn't think there's anything completely abnormal here. Tb counts are relatively the same, current is the same, as well as RSD duals. I used to have a consistent 7-9% Bead CV% with my old cone that would change depending on how well I mixed the bottle of beads before aliquot. Whereas the new cone we received, gives consistently 9-11% CV, also depending on how well I mix the bottle.

The MakeUp gas (0.8) seems high to me however, and although it's consistent between new and old cones, I wonder if you could reduce the CV% in general by checking for leaks in your Spray Chamber (old o-rings?), or clogs at the tip of your Nebulizer. If you get that Neb gas down below 0.6, I'd expect you'll see lower CV%, but I wonder if you'll also see a drop in current and increase in Tb dual-counts.

Best wishes,
Matthew
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desireeBCRT

Contributor

Posts: 23

Joined: Mon Jan 06, 2014 11:56 am

Post Mon Sep 16, 2019 9:43 am

Re: "New" cones from fluidigm

Hi Mark,

I also recently got a new skimmer/reducer cone for my Helios, which upon installing and tuning resulted in a current of 8 and a bead CV of 20%. There were no obvious signs of damage. I got a replacement from Fluidigm after complaining and sending in the test results, but still have to test this new one.
The skimmer came with an engraving, but others have the same and are fine.

Best
Desiree
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mleipold

Guru

Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon Sep 16, 2019 2:18 pm

Re: "New" cones from fluidigm

Hi Mark,

To confirm: you have a Cytof2, correct? I had been incorrectly thinking it was a Helios, but Matthew's comment about the 0.8 MG made me go back and reread your original posts about the Cytof1/2 cone (as well as the software screenshot not being Helios)

If so, then your spray chamber should still be glass and Matthew's spray chamber o-rings comment doesn't really apply.

I would agree, the tip of the skimmer in your first post looks suspiciously dark, like it's been used.

Bead CV also has to do with the width of the cone orifice. As it ages, it widens and your CV can increase. Additionally, the orifice can go from circular to slightly non-circular.

How do the orifices of the two skimmers compare?


Mike
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markyyy

Participant

Posts: 15

Joined: Wed Jul 26, 2017 4:09 pm

Post Tue Sep 17, 2019 2:49 pm

Re: "New" cones from fluidigm

Hi all,

Thank you for the responses. Yes, we are working with a CyTOF2 so I am not concerned over our gas numbers. I have yet to look at the two cones side by side under a microscope. I will do that. Mike, considering the fact that the two cones are so close in tuning specs, would you expect me to see a major difference? Again, given the fact that the tuning specs are so well within acceptable for our old cone would you expect to see a difference between it and a completely brand new one?

At this point I am relieved to hear that the engraving is normal, but I still consider the amount of wear on the "new" cone - both in terms of discoloration and markings from the removal tool - to be more than should be expected on a part that only needs to be tested once for a short period of time. I think I will send it back and hope that a replacement is in better condition.
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mleipold

Guru

Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Tue Sep 17, 2019 2:52 pm

Re: "New" cones from fluidigm

Hi Mark,

I wouldn't necessarily expect a major difference between the old cone and the "new" cone, if the values are similar.

That said, it sounds a lot higher than a "new" cone should be. Back when we had CyTOF1 instruments, a new skimmer would typically start out around Current = 4. If your old cone is several months old and is around Current = 6.5-7, that's one thing.

But I don't see how an *actual* new cone could possibly be Current = 6.5 without there being something wrong (either with the cone, or something with your instrument).


Mike
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GregBehbehani

Master

Posts: 85

Joined: Tue Apr 12, 2016 10:17 pm

Location: The Ohio State University, Columbus, Ohio

Post Tue Sep 17, 2019 3:22 pm

Re: "New" cones from fluidigm

Hi Mark and Mike,

As I said previously, I'm not very familiar with a CyTOF2, but I'm pretty sure it has a narrower injector than a CyTOF1. I think on the newer machines (esp. Helios), the "current" voltage needs to be higher because the ions are moving faster (due to the narrower injector) and thus have less time to be focused by the current voltage. In our experience, the optimal current increases pretty quickly once you start using a new set of cones, as the surface gets a layer of oxide that acts to insulate it a bit (which regular cleaning doesn't ever completely remove).

All of this is just to say that a current of 6.5V wouldn't be terribly surprising to me for a cone used for several hours (at least on a Helios). So unless your old cones were clearly lower than this when they were a day or two old (you should be able to look back at your tuning log to see), I wouldn't get too worried about that number. Although if you're not happy with the cone, it seems reasonable for you to send it back for a replacement.

best,

Greg
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