FAQ  •  Register  •  Login

Blocked nebuliser / nebulizer

<<

jimbomahoney

Master

Posts: 60

Joined: Wed Feb 27, 2019 11:21 am

Post Fri May 17, 2019 8:03 am

Blocked nebuliser / nebulizer

Hi all,

Does anyone have advice on how to clear a blocked / clogged nebuliser?

We were running what looked like (by eye) lovely clear samples (mouse splenocytes) filtered and diluted to achieve ~ 300 events/sec.

I managed to clear the clogs a couple of times by flushing with 10% decon 90 (as per Fluidigm instructions) and carry on running.

However, every ~10 mins the freshly cleaned one would clog and now both nebulisers are still blocked after soaking overnight in 10% decon 90.

I cannot flush any liquid through from the back (i.e. the sample input end).

I can flush liquid through the gas input side.

For the moment, I'm just leaving them to soak in Decon 90 until I find a way to clear them, or buy new ones.

I've read about a mysterious (to me) Pearl Cleaner, but unsure exactly what it does?
<<

Marjolyn

Participant

Posts: 7

Joined: Tue Sep 15, 2015 6:02 pm

Location: Leiden, The Netherlands

Post Fri May 17, 2019 10:46 am

Re: Blocked nebuliser / nebulizer

Hi Jim,

We have multiple ways that we unclog nebulisers.

- 1st we try to flush the inner capillary with hot MQ water
- when dis doesn't work, we try the hair method (without the root)
- when the clog is still really stuck ik will help to take up hot MQ water in the outer part of the capillary to smoothen up the clog.
- try again with hot MQ for the inner cappilary

You can check under the microscoop if the inner capillary is clean and has a smooth wall. When this is not the case, cells can easily stick to this walls and again form new clogs.
- when this occurs we wash the nebuliser with 1:1 MeOH/MQ and let the CyTOF run with the nebuliser held in the 50 ml tube (never into the spraychamber)
This solution will really strip of all kinds of cell debris and small clogs.

Hope this helps.
Good luck.
Marjolijn
<<

DMcDonald

Participant

Posts: 11

Joined: Thu Nov 26, 2015 10:14 am

Post Fri May 17, 2019 11:12 am

Re: Blocked nebuliser / nebulizer

Hi James,

I can hopefully share a few tips that so far have cleared out even the most stubborn blockages.

Take a 25mL luer lock syringe (the standard fluidigm supplied one is too small to have any umph) with some tubing attached via a barbed luer lock adaptor (cole palmer). Slip this over the back end of the nebuliser like you normally would. I then put the business end of the nebuliser through the rack of a P1000 tip box that's filled with HOT 10% contrad (80 oC ish in a water bath). After letting the nebulisers warm up I pull hard on the back of the syringe to create a big vacuum and wedge the syringe in place. I leave this overnight. Often this is enough to clear the nebuliser if the blockage was biological or salt (from someone not following protocol). I'd say about 50% of the time though really bad blockages are from microplastics building up in the tip. this is less of an issue with the push fit fast fittings than the older screw in ones but we still get them from some tubes/samples. These I clear by repeating the above with acetone but using a smaller vessel and not heating so much. After these fully flush with water and fully dry with argon/clean compressed air. I also check they're completely clears on an inverted microscope and that the operating pressure once reconnected is back to normal.

It's important too to say that I never push on the syringe to flush, only ever pull. And if you need to release the vacuum formed do so by opening the luer lock fitting.

I'm also interested to hear about the pearl though to see if it's of any use.

Hope that helps,
David
<<

EHaasDFCI

Participant

Posts: 19

Joined: Thu Jun 15, 2017 5:34 pm

Post Fri May 17, 2019 1:08 pm

Re: Blocked nebuliser / nebulizer

Hi James,

We have used the pearl nebulizer cleaner before with some success. However, as mentioned above, for super stubborn clogs, the best bet is a hot 10% contrad bath. I would heat that up, leave the nebulizers soak in the warm water, then leave them soak over the weekend and possibly even next week. The warm solution is the only way to go for really bad clogs. From personal experience, I would recommend against trying to push the clog through with anything, it's just a quick way to a broken nebulizer :lol:
<<

mleipold

Guru

Posts: 1778

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri May 17, 2019 3:06 pm

Re: Blocked nebuliser / nebulizer

Hi all,

As James linked previously,this is the Meinhard Pearl Nebulizer Cleaner tool: https://www.meinhard.com/index.cfm/prod ... lizers.cfm

We (and several other labs) have used it with pretty good success. The main advantages of using the Pearl tool are that you can make a tight seal around the nebulizer, which then allows you to use the plunger part to forcefully backflush fluid through both the sample capillary (to drip out the sample port stem) and the gas chamber (to drip out the gas side port). Basically, you take out the plunger, unscrew the black cap, then insert the nebulizer, screw in the black cap until just barely tight, pour cleaner in the top chamber, then use the plunger to push it back through the nebulizer. You then unscrew the black cap and remove the nebulizer before you pull the white plunger back out.

The Pearl cleaner we use is 40mL of 5% citranox and 10mL of methanol. A few flushes of that will clean out most clogs, and do it faster than nitric.

The disadvantages of the Pearl tool are:
1. You can break the nebulizer internal capillary. This is a certain amount of risk you take using it. Basically, the forceful backflushing on occasion will break the capillary: I think it's flow forces causing it to wiggle back and forth, and snap. It doesn't happen often, but it does happen.
2. Since the nebulizers are hand-made (last I heard, at least), there's a small variance in the outer diameter of the nebulizer gas chamber. Therefore, occasionally you'll get a nebulizer that's too fat to fit in the hole, and similarly occasionally you'll get one that's a little too narrow to form a sufficient seal to actually flush the nebulizer capillary....instead, it will just spill around the nebulizer rather than really going *through* the nebulizer.

However: the Pearl nebulizer flushing does require there to be *flow*. If the sample capillary is completely blocked, it won't clean.

Generally, if the capillary is completely blocked (with organic matter), then the only thing I've had good success with is leaving it inverted in concentrated nitric for a few days. There, even if it's completely blocked, capillary action and diffusion will *eventually* get nitric up to the clog so it can dissolve (or at least loosen) it.


Mike
<<

jimbomahoney

Master

Posts: 60

Joined: Wed Feb 27, 2019 11:21 am

Post Mon May 20, 2019 12:19 pm

Re: Blocked nebuliser / nebulizer

Hi all,

Thanks for your tips.

I left both nebulisers in FACS tubes filled with isopropanol over the weekend.

This morning, I managed to get liquid up through them, and once there is SOME flow, the blockages loosened and got sucked up the capillary.

They are now both clear, so I've flushed them with 18M water and left them running wash solution from the PSI into a FACS tube.

Spray looks nice and even now, so fingers crossed!

:D

Key lessons from my perspective are:

1) Agree with David - only "pull" or "suck" through the nebuliser sample capillary. It gets smaller towards the output side, so pushing air or liquid down will only jam the clog more. We have some lovely "stylus cleaners" from BD. They fit through the "input" side of the sample capillary easily, but frustratingly get stuck about 10mm from the "output" side. Otherwise they would be perfect for "forcing" a blockage. I estimate that they are ~150 µm thick and that the output end of the nebuliser sample capillary is ~100 µm.

2) The "hair method" is interesting - it's possible to fit a piece of my hair into the output side, but (unlike the BD stylus) it's not stiff enough to push the blockage free.

3) In this instance, I didn't need any heated solution or nitric acid. The blockages were both brown in colour and visible ~ 10mm from the output side of the nebuliser.

4) For future blockages, I think I will adapt David's advice and leave syringers with vacuum applied and the nebulisers in FACS tubes overnight / over a weekend.
<<

mleipold

Guru

Posts: 1778

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon May 20, 2019 2:48 pm

Re: Blocked nebuliser / nebulizer

Hi James,

I'm glad that your nebulizers have cleared.

FYI: if you're just doing a soak in nitric or something, I've found that the 2mL collection tubes that you use for spin filters (ike in antibody conjugation) are *just* the right depth. The gas port side-arm is at the right height to suspend the tip of the nebulizer about 2mm off the bottom of the tube.

This has the advantage of minimizing the volume of soak you need, which is especially important when working with something like concentrated nitric acid (the nitric doesn't eat the tube plastic on any reasonable timescale......not even days).


Mike
<<

jimbomahoney

Master

Posts: 60

Joined: Wed Feb 27, 2019 11:21 am

Post Mon Jul 29, 2019 1:13 pm

Re: Blocked nebuliser / nebulizer

Just an update after getting some more clogs:

I'm loving the Pearl Nebuliser Cleaner, which I ordered after experiencing the first clogs that led to the creation of this post.

https://www.meinhard.com/index.cfm/prod ... lizers.cfm

(No commercial interest!)

This is so quick and easy to use - I'd go as far as to suggest that Fluidigm should supply one of these with every system (I'll suggest to my local rep).

Return to Introduction to mass cytometry

Who is online

Users browsing this forum: No registered users and 2 guests