blocked nebulisers

[rgrenfell]

Really struggling with blocked nebulisers at the moment.
Running DIP samples, from several users, but they are all tending to block the nebuliser, right at the tip
I'm doing about 7-8 samples a day, and probably getting 3-4 properly blocked nebulisers in that time.
concentration is somewhere between 500k and 1 million per ml, filtered and running n CAS. 250-450 eps is what I'm seeing

the blockage is always a whiteish solid right near the very end of the nebuliser. Water doesnt clear it, acid doesnt clear it, the only thing I have found is soakingin Contrad for 30 mins, with a syringe pulling up on it.

Really frustrating, it doesnt seem to be any batch of reagent, users are washing as per the protocol, but it just keep blocking

Photo of the blockage attached
Any ideas?

Thanks
Richard

[rgrenfell]

cant seem to attached photos, sorry

[mleipold]

Hi Richard,

Your files might be too big to attach using the "Upload Attachment" section. You can always upload them to another site like Dropbox or Google Drive and post the link.

By "DIP samples", do you mean samples stained with the MDIPA lyo cocktail? If so, what kind of samples are they (PBMC, WB, etc)? Or are they some other kind of sample type entirely?


You mention a whitish solid: is it appearing in the internal capillary where the sample is introduced? Or also covering the gas opening? Or is it covering the entire tip (including the exterior surface)?


Mike

[vscarfone]

Hi Richard,

I have found hot contrad to work well. I heat it for about 15-20 seconds in the microwave and pull it through with a syringe (as you would normally clean). Depending on how bad the clog is, you can soak, then rinse with water and check under the scope.
I have also been testing BD's Aria (cell sorter) sample line filters as they fit right on the Helios sample line. I have been using a 50um filter, which helps reduce clogging tremendously. I haven't seen much metal contamination from it either.

Good luck,
Vanessa

[rgrenfell]

https://drive.google.com/file/d/1_5uCkM ... sp=sharing

A link to the photo of the nebuliser (I hope)

blockage is only ever in the sample capillary int he nebuliser, never in the gas part

These are PBMC with the Fluidigm MDIPA staining, some of them have additional antibodies, but most dont.
Thanks
Richard

[mleipold]

Hi Richard,

I wasn't able to access the Drive link: I had to request permission.


Mike

[Chowduck]

Hi Richard,

As a clog is forming you should expect to see the PSI pressure increase and the event rate decrease. Stopping the run immediately at this time will make dislodging the debris and cleaning much easier before a complete blockage is formed.

You might consider using a syringe with luer adapter a few times a day to backflush from the grounding nut to a waste tube inside the PSI in order to dislodge debris in the chokepoint of the grounding nut and upstream tubing.

The Helios manual recommends leaving at least 50uL in your sample tube to avoid neb clogs as the dregs will have more debris and aggregates.

Filtering the sample through 35 micron nitex immediately prior to loading will help to remove debris and aggregates.

As a last resort I have used a strand of straight human hair to delicately poke at the clog but this is very risky and has a good chance of breaking the neb.

Greg Chang

[GlassExpansionInc]

Hi Richard,

Glass Expansion has designed a nebulizer for single-cell analysis and we have a model specifically designed to suit the CyTOF and Helios instruments, product number C21-1-UFT02. Glass Expansion design feature our unique VitriCone sample capillary. Non-Glass Expansion designs typically use a hand-drawn glass sample capillary. With hand-drawn sample capillaries, the internal diameter can vary, preventing a laminar nebulizer flow and creating points where particulates may lodge.

The Glass Expansion VitriCone sample capillary is entirely different. It is machined from a thick-walled glass capillary, providing a highly reproducible geometry and constant internal diameter – from sample inlet to tip. The constant diameter, large bore sample channel minimizes blockages from sample build-up commonly experienced with biological samples.

Our Eluo nebulizer cleaning tool, product number 70-Eluo, is designed to efficiently and safely deliver a cleaning solution through the nebulizer capillary to dislodge particle build-up and thoroughly clean the nebulizer. Here is a video on how to use the Eluo to properly clean your nebulizer: https://youtu.be/587QFobdQgE

Feel free to contact me directly, at rbrennan@geicp.com, if you have any specific questions about our nebulizer or maintenance procedures.

Sincerely,
Ryan Brennan

President, Glass Expansion Inc.
Pocasset, MA 02559 USA

[rgrenfell]

I hope the permissions are set ok now

https://drive.google.com/file/d/1_5uCkM ... p=drivesdk

Richard

[mleipold]

Hi Richard,

Thanks, yes I can see the picture now. To me, this looks like standard brownish cell material clogging the nebulizer internal sample capillary, rather than some sort of weird salt build up.

Unfortunately, I don't have much to suggest beyond what other people have said. I will say, in my opinion, your 250-450 eps rate is on the high side: I know that Fluidigm claims that up to 500 eps is OK, but we have noticed over the years that there is a substantial increase in clogging at higher rates. As a result, our target event rate is 200-300 eps, and we do not run above 350 eps even with a Supersampler (which seems to clog a bit less than the PSI, probably due to better mixing causing better dispersion and less clumping during acquisition).

(and beyond that, in our experience the doublet rate increases significantly above 350 eps anyway, especially if you have the longer EL due to a CYTOF2 injector or a WB injector)

I would also ask: do you know if your users are using Benzonase during thaw? I know that clumps can happen even after benzonase+RPMI thaw/washes (taking them out of media into buffer, after antibody addition, etc), but that is one major area where we have to remind customers.


Mike