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Persistent contamination and Nebulizer issues

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mds4z

Participant

Posts: 8

Joined: Tue Dec 03, 2013 6:23 pm

Post Thu Jan 22, 2015 4:58 pm

Persistent contamination and Nebulizer issues

Hi Everyone,

We’ve been experiencing 3 different consistent problems with our CyTOF 2:

1.Persistent Iodine contamination – Fluidigm’s solution is always to change the sample line, change the nebulizer, change the tuning solution, water etc etc. We have done that and that still persists. Has anyone experienced this and if so, has anyone identified the source?

2. Barium events coinciding with Iridium events – We’ve noticed in ALL samples that we’ve run that there is consistently with almost all events identified as a cell event by Iridium, an equivalent puff that coincides with Barium. The samples have had nothing done to them (no special drug treatments or imaging). This has happened with ALL users samples from different labs and all the users all use Fluidigm’s Fix/Perm reagents, buffers and water and we are seeing this consistent Barium puff. We could almost use it instead of Iridium as the intercalator to identify cell events instead of Iridium!

3. Nebulizers – We’ve been averaging 1 run a nebulizer recently before some kind of trouble. We filter all our samples immediately before running them, and then eventually we see dramatically decreased event rate, signals drop. Short of seeing a drip, how does everyone identify that there may be some kind of partial clog? We would expect to see leaking at one of the points in the sample line caused by back pressure from a clog but recently we experienced what seems to be a partial clog without any dripping… Do people clean their glassware with Citranox?

The frustrating thing is that Fluidigm always logs in, makes us clean for almost an hour and then eventually tells us to change our nebulizer and/or sample line and we can just keep changing them every single run, that’s impractical and expensive and wastes time. Any insight would be appreciated!

Thanks,
Claude
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri Jan 23, 2015 4:38 pm

Re: Persistent contamination and Nebulizer issues

Hi MIchael,

1. When you say Iodine contamination, do you mean a persistent streak/track at Mass 127, or light, intermittent speckles?

I've heard from at least one source that the glass of the syringe in the syringe pump for a CyTOFv2, as shipped, could have traces of something on it. I heard specifically about lead, but it's possible iodine would be another contaminant. If so, then it would contaminate everything downstream from the glass syringe.

Thankfully, I've heard that a soak in nitric acid does a good job of stripping out the contamination (at least for lead). Make sure you use an ultrapure nitric acid like Fisher Optima grade (A-467-500). Be careful handling it.

I also remember years ago having a bottle of Tuning solution that was contaminated with iodine. Again, nitric acid did a good job washing it out of the machine. For a CyTOFv1, you could just put it in the plastic syringe on the syringe pump and drive it through the entire system. For a CyTOFv2 pulling from a reservoir bottle, you would have to put the source line in a reservoir bottle of 3% nitric to do the comparable wash.


2. Barium can be really really tricky to get rid of entirely. At the HIMC, we've accepted that most samples are going to have at least a trace of barium associated with the cells. However, some samples can be much worse than others. You might check with your users to make sure that they're using ultrapure reagents like Rockland's 10x PBS, rather than standard Gibco cell culture PBS....I know I've had that issue with some of my customers.

Barium can also linger in the machine; again, 3% nitric seems to do a better job of stripping it out than just Wash solution alone does.


3. Nebulizer clogs: generally speaking, if your connections are tight, you won't see dripping. What you're likely to see instead is a dramatic decrease in cell event rate, then all of a sudden a bunch of cells going through as the partial clog bursts through. Occasionally, if this clog gets big enough, the burst through will shut down the plasma (large droplets or even a stream, rather than fine spray of droplets going into the plasma overcools it and the plasma guidelines shut it down).

We store our nebulizers filled with 5% citranox, submerged in citranox. Before each day's use, we backflush the nebulizer twice through each port using a 3mL syringe and some tubing. We then forward-flush through the gas port to make sure the stream is coaxial, and not spraying in two streams or something. We then backflush twice with MilliQ water, then forward flush with MilliQ water to again check how coaxial the stream is. At the end of the day, we remove the nebulizer, and backflush each port at least twice in citranox before filling it with citranox and submerging it back in citranox for storage.

If the nebulizer *is* spraying funny or backflushing slowly (both signs of a clog before you even put it in the machine), then we partially fill by backflushing with and submerge in concentrated nitric acid (which won't harm glass). Again, the high-purity nitric mentioned above. We've found that the 2mL microcentrifuge tubes that come with 500uL spin filters are just the right length to use for soaking in a minimal amount of nitric: the gas port arm on the nebulizer keeps the tip a couple millimeters from the bottom of the tube.


You're more likely to see leaks when there's a clog in the nebulizer tubing, as back pressure can build up more. A clog there will also slow down your event rate; eventually, it will clog completely. On a CyTOFv1, the back pressure generated would pop the tubing off the friction fitting end of the syringe in the syringe pump. On a CyTOFv2, I've heard that it'll cause dripping at the glass syringe.

Clogs in nebulizers and in nebulizer lines kind of enter a vicious circle: any time you have a clog in either one, you slow down sample flow and accumulate debris. So a clog in one can help cause a clog in the other one.

I should say: since they're supplied at a fixed concentration, you can use the calibration beads to help diagnose whether there's a clog. If you're developing a clog, your bead event rate will also go down.


How often are you running Wash solution? Between every few samples, or just at the end of the day? Also, the type of sample will make a difference: well-prepared PBMC samples are usually fine. But whole blood samples require greater dilution and more frequent cleaning, as they have more sticky crap in them. Some cell lines are fine, but we've had terrible luck with Raji cells, no matter how much we dilute them.


Mike

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