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Ba contam in gut biopsy samples

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MattCochran

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Joined: Fri May 30, 2014 3:34 pm

Post Thu Aug 24, 2017 3:25 pm

Ba contam in gut biopsy samples

Hello fellow CyTOFers. I have a couple questions regarding an ongoing project that has developed some contamination issues along the way. In the study, we have both gut biopsies and PBMC in matched pairs. Midway through a large cohort of samples we began having extensive Ba contamination issues with the gut samples only, with no increased contamination in the PBMC. As far as we can tell, there were no changes in the processing of the samples, in fact the contamination started by showing up in two but not the third of three samples all processed and run on the same day using the same reagents and has been prevalent ever since that point. We went on an extensive hunt for the source, testing the existing and new batches of reagents by running them in solution as well as with/without on cells. We’ve found a few reagents that show higher than we’d like Ba levels in solution but we’ve not found one culprit that we can point to as the problem.

From this I have a few questions:

1. Has anyone else used gut biopsy samples that might have thoughts? Are gut samples more prone to Ba contamination and we possible hit a bunch of “naturally” contaminated samples in a row?
2. Is it possible that a room/hood/space becomes contaminated and then can pass that contamination to samples that are processed there?
3. Has anyone seen a situation in which a few mild Ba signal reagents have combined to create massive contamination in the samples processed with them?
4. Is there a way to translate the levels of Ba signal in the reagents run as solution to expected levels of contamination in the cells?

Thanks for any help you might be able to offer.

Matt
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dahern

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Joined: Tue Mar 31, 2015 3:45 pm

Post Thu Aug 24, 2017 5:05 pm

Re: Ba contam in gut biopsy samples

HI Matt,

Just a thought but do you use the inside of a 1ml syringe to 'mash' the biopsie through a cell strainer at any point? I know some people do with spleens etc. We found that certain makes of syringe when used like that led to a massive barium contamination.
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FlowjoVA

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Joined: Tue May 24, 2016 4:49 pm

Post Thu Aug 24, 2017 5:38 pm

Re: Ba contam in gut biopsy samples

Hi Matt:
Is the patient the source? Recent Barium enema? The fact it is in some patient samples but not others leads me to think it is patient associated and certainly GI patients can have seen barium in diagnostic procedures???
Just a thought...
Joanne
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MattCochran

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Joined: Fri May 30, 2014 3:34 pm

Post Thu Aug 24, 2017 6:25 pm

Re: Ba contam in gut biopsy samples

Two great responses already. Thank you.

The syringe mashing is something we haven't tested directly but we don't think that process has changed. We do plan to try and test this directly though.

We are definitely wondering if it is in fact patient specific. We did test the enema and saw some Ba signal but didn't think it was enough to cause these issues. We didn't know of other potential sources specific to GI patients, but if you do, I would love to hear some examples we could take back to the MDs.

This board is great. We really appreciate the help.
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GregBehbehani

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Posts: 85

Joined: Tue Apr 12, 2016 10:17 pm

Location: The Ohio State University, Columbus, Ohio

Post Fri Aug 25, 2017 4:42 am

Re: Ba contam in gut biopsy samples

Hi Matt,

I would agree with Joanne's suggestion of barium contrast, I would strongly suspect that this is the most likely cause as barium contrast imaging would frequently be performed before a colonoscopy and biopsy. (You may also want to make sure you don't have iodine contamination.)

I would also point out that heavy metals can stick to the the tubing in the CyTOF (the sample loops of the CyTOF1 and 2 and in the PSI flow sensor on a Helios) and lead to persistent background signal for quite a while after the contaminated sample was run. The barium does seem to get "picked up" by the cells to some extent when this carryover occurs. I suspect that if you thoroughly clean your fluidics, you can get rid of the Barium (at least until the next time you get a patient sample post-barium enema).

Let us know what you figure out.

best,

Greg
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mleipold

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Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon Aug 28, 2017 8:54 pm

Re: Ba contam in gut biopsy samples

Hi Matt,

To chime in a bit on Greg's point about cleaning the fluidics: I often find that 3% nitric (or, running Tuning solution which has 2% nitric) does a better job of cleaning Barium out of the fluidics than the Wash solution does. Since you have Tuning solution on hand, you can just run that until the Barium gets under control.

When you say that you did " test the enema and saw some Ba signal", how did you test that? Remember, Barium salts are generally really insoluble in pH neutral water (at 20C, BaSO4: ~2.5 ug/mL, Ksp = 1.0842 × 10−10). Therefore, if you took some of the enema solution, dropped it in water, then filtered it, very little of the Barium would likely have dissolved into your signal. If you repeat the experiment but use 3% nitric instead of using water, I'll wager you'll get a *much* higher signal.

Finally: remember, anything that gets ingested could potentially wind up in a gut biopsy sample. So, if you start seeing Bi209 contamination, your donor might have recently had something like PeptoBismol.......


Mike
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cualera

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Joined: Wed Aug 02, 2017 6:16 pm

Post Fri Jan 19, 2018 7:15 pm

Re: Ba contam in gut biopsy samples

Hi everyone,
I work with gut biopsies and most of them have high levels of Barium. I would like to add that we found PFA ampoules also a source of Ba contamination.
I am wondering what could be the criteria to throw a sample out of the analysis if we observe Ba contamination.
Anybody has information about serious spillover of Ba or Ba will stay in 138 without disturbing other channels?
thanks
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taxkourel

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Posts: 14

Joined: Sun Sep 03, 2017 3:46 pm

Post Fri Jan 19, 2018 7:59 pm

Re: Ba contam in gut biopsy samples

cualera wrote:Hi everyone,
I work with gut biopsies and most of them have high levels of Barium. I would like to add that we found PFA ampoules also a source of Ba contamination.
I am wondering what could be the criteria to throw a sample out of the analysis if we observe Ba contamination.
Anybody has information about serious spillover of Ba or Ba will stay in 138 without disturbing other channels?
thanks

This might be reaching and totally wrong but if these are human samples and you happen to have clinical history make sure that they dont come from patients that had diagnostic procedures that included barium ingestion. I think i have read something similar for gadolinium signal in pts getting MRIs with Gd contrast.
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GregBehbehani

Master

Posts: 85

Joined: Tue Apr 12, 2016 10:17 pm

Location: The Ohio State University, Columbus, Ohio

Post Fri Jan 19, 2018 10:45 pm

Re: Ba contam in gut biopsy samples

Hi,

With regard to the criteria to throw out a sample, it would simply be if any of the channels you care about are being altered by the Ba contamination. In general, you should be able to tolerate contamination of up to a few hundred counts per cell event if it's happening on a channel that you're not using for an antibody. Above that, it would just depend on how much M+1 spillover you're getting into the 139 channel (if you're using it) and how much M+16 you're seeing in 153 and 154. In general, once you get into thousands of counts per cell, I would think you spillovers would be too high to get reliable data. The detector should be fine up to many thousands of counts (unless your contamination is in absolutely every cell you run), so the real limitation would be the spillovers that you should be able to readily evaluate.

best of luck,

Greg
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mleipold

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Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Sat Jan 20, 2018 12:07 am

Re: Ba contam in gut biopsy samples

Taxiarchis: the Gd in MRI samples was mentioned In Chevrier et al 2017: viewtopic.php?f=10&t=707&p=2147&hilit=gadolinium+contamination#p2147

"Gadolinium Contamination Test
Some patients were scanned during diagnosis with magnetic resonance imaging using a gadolinium-containing contrast agent. To identify samples positive for gadolinium, a small aliquot of each sample was collected after fixation and analyzed individually in the mass cytometer."
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