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Super high barium contamination?

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AdeebR

Grand master

Posts: 169

Joined: Thu Mar 13, 2014 5:58 pm

Location: NYC

Post Tue Nov 04, 2014 12:22 am

Super high barium contamination?

Hello CyTOFers,

My CyTOF is currently somewhat unhappy after running some clinical tumor samples. I ran one sample last Thursday, after which I had massive barium contamination of the instrument (Ba138 duals well over 1 million while running diH20). It was very difficult to get rid of, but after troubleshooting with tech support and alternating wash solution and water for several hours I finally got my Ba background back down to normal levels. Today I ran another tumor sample (different patient, different kind of tumor) and now I'm back in the same situation - sky high barium levels while running diH20.

So this brings me to my 2 questions:

1) How best to clean the instrument? I'm surprised at how "sticky" this contamination seems to be. I noticed that the levels would periodically start to come down while running diH20, but another round of wash solution would cause a new spike in the levels, which makes it seem like more was being liberated from the tubing with each wash.

2) What might be the source? I've seen samples before that were washed in Ba-containing buffers and in those cases it seemed like all the cells were somewhat high for barium. The situation in this case seems a little more unusual: I wasn't specifically collecting data for the 138Ba channel, but I do see high background staining in In115, Xe131, Cs133 Ce140 (all channels that did not contain antibody and also in Sm154, where Ba138 oxides would presumably appear (see attached PDF). However, most of this background is present in the non-CD45, DNA-negative fraction. Have others seen anything like this? Any thoughts on what's going on and how to avoid it in the future? I suspect it's something originating from the pathology suite where the tissues are being collected from but I'm not sure.
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Background_contamination.pdf
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Adeeb Rahman
Icahn School of Medicine at Mount Sinai, NYC
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Tue Nov 04, 2014 12:56 am

Re: Super high barium contamination?

Hi Adeeb,

I'm a little unsure that Ba is your only problem in this case.

Ba is 130-138, with Ba138 being 72% of the natural abundance. There shouldn't be any background staining in In115 due to that.

When you were running these samples, what did the In115 channel look like as the cells were going by? Was it streaky like there was a ton of background/free antibody? If so, it's possible that you have a Tin (Sn) contamination: we've run into that a couple times. Tin is 112-124; there's Sn115, albeit at only 0.34%. Sn118 is a good channel to monitor: it's 24% of natural Sn, and is the only 118 mass.

For reference: Sn is at lower mass than (to the left of) the Xe speckles we see as impurities in the argon gas. Ba138 is the extreme right edge of the Xe speckles.

From the gating in your attachment, "non-CD45, DNA-negative" would be the bottom row?


We've never been able to completely figure out where the tin comes from, from time to time. Once it appeared to be in magnetic enrichment beads we used on our samples. Another time, it appeared to be in the water someone used to wash and resuspend their cells.


Regardless: we've found that for removing both Sn and Ba, 3% nitric appears to work a bit better than Wash solution (it also does a better job for Cd, Pt, and Pd). Ba strips out fairly fast, but Sn seems to linger....we've run 3% nitric from the syringe pump to make sure that we're getting the entire system.

One other possibility: I haven't tried this, but I know that some elements have differing affinities for the tubing and other parts of the CyTOF. For example, Ir has a lower affinity for the system than Tb159 or Tm169 does. So, occasionally, when I have particularly bad Ir residue, I'll run a bunch of tuning solution: the Tb and Tm help kick off the residual Ir (as fast or faster than Wash), and then Wash solution or 3% nitric to remove the tuning lanthanides.


Regarding the sample prep form the pathology suite: if they're making PBS or other buffers up in bottles that have gone through a departmental dishwash, you'll never get rid of the Ba issue. I remember early on, DVS and the Nolan Lab discovered that issue, and I think that no matter how much they rinsed out the bottles or even washed with acid, the Ba lingered and kept recontaminating their buffers.


You could try getting a sample of each of their buffers, and running them in liquid mode to check for contaminants.....might be the fastest way to find the culprit.


mike
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AdeebR

Grand master

Posts: 169

Joined: Thu Mar 13, 2014 5:58 pm

Location: NYC

Post Tue Nov 04, 2014 2:44 pm

Re: Super high barium contamination?

Thanks Mike. I was also confused by the signal in the In115 channel. I was wondering if it was just some sort of abundance sensitivity issue making the CyTOF go "crazy" (technical term)

There was actually very little streaking while running the samples - the events that I was seeing in the In115-Ce140 channels seems to appear as very distinct and well-separated ion clouds, which were typically negative for Ir191/193 signal.

The tin suggestion is interesting and seems like it makes sense, but what's strange is that now, while trying to clean and run diH20 the only contamination that I'm seeing in the ToF window with a mass range from 79-212 is Ba (with only one major pronounced peak at 138).
Adeeb Rahman
Icahn School of Medicine at Mount Sinai, NYC

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