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Unusual staining in 140-150 range

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nkhanbham

Master

Posts: 53

Joined: Wed Feb 25, 2015 3:03 pm

Post Wed Jun 21, 2017 6:06 pm

Unusual staining in 140-150 range

Dear all,
Has anyone experienced the following unusual staining? We are getting events that are Iridium +ve (and Cisplatin -ve) but with intermediate levels of 140Ce signal (see attachment). These "unknown" events show very high positivity across 140-150 range, which includes markers that cannot all be co-expressed such as CD19, CD4, CD11b, CD34. The events are not EQ beads stuck to cells, as we still see the abnormal staining in samples run without beads added. In the example shown it is only 0.2% but we see upto 5% in some samples. This has been observed using blood and bone marrow and using three different independent antibody panels.
I welcome any feedback.
Thanks,
Naeem
Attachments
Unusual CyTOF staining Bham RPT 2017.pptx
(342.75 KiB) Downloaded 200 times
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mleipold

Guru

Posts: 2256

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed Jun 21, 2017 6:12 pm

Re: Unusual staining in 140-150 range

Hi Naeem,

Are you using PFA (16% stock, brown glass ampule, usually from EMS though it might be repackaged)?

If so, try filtering it and see if that makes a difference: some people have reported Ln-containing particulates in PFA stocks that vary by lot (and even a bit by ampule within a lot).

See:
viewtopic.php?f=4&t=713&p=2173&hilit=PFA#p2173
viewtopic.php?f=7&t=146


Mike
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AdeebR

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Location: NYC

Post Thu Jun 22, 2017 2:13 am

Re: Unusual staining in 140-150 range

Also, have you tried filtering your antibody cocktail through a 0.1micron spin filter?
Adeeb Rahman
Icahn School of Medicine at Mount Sinai, NYC
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nkhanbham

Master

Posts: 53

Joined: Wed Feb 25, 2015 3:03 pm

Post Thu Jun 22, 2017 4:39 pm

Re: Unusual staining in 140-150 range

Thanks for the quick replies.

I am using Fix Perm buffers from Fluidigm. These are all in plastic bottles. I have thought about filtering the antibody cocktails. I have never come across filters for liquids at such tiny volumes (<20ul). Where does one get these from?
Naeem
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GregBehbehani

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Posts: 80

Joined: Tue Apr 12, 2016 10:17 pm

Location: The Ohio State University, Columbus, Ohio

Post Thu Jun 22, 2017 7:49 pm

Re: Unusual staining in 140-150 range

Hi Naeem,

It looks most consistent with metal contamination. I notice there may be similar events in your data that are not Ir positive. Did you use magnetic beads on your samples? This could be a source of natural Nd, which could have other rare earth metals (like Ce) with it. That would fit the pattern you're seeing.

I have also seen non-specific staining in which certain cell types (likely eosinophils) will become sticky to essentially all antibodies. Adeeb's group published a paper about this (viewtopic.php?f=10&t=465&p=1477&hilit=Adeeb+eosinophil+cytometry#p1477). If that were the case though, I would expect that antibodies other than just the 140-150 range would also be positive on these events.

I would re-run your sample while recording the entire mass range so that you can get a plot of every mass present on these events and an accurate median for each. Typically for metal contamination, once you know the precise isotopic distribution the culprit can be figured out from the relative natural isotopic abundance of the elements in that mass range.

It would also be a good idea to run your solutions (particularly the ones you used on your un-stained cells) to see if the metal can be found in any of these. You could also run a bit of the cells without any antibody staining (just stained with Ir) to see if this is present before staining.

Let us know if you figure it out.

best,

Greg
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nkhanbham

Master

Posts: 53

Joined: Wed Feb 25, 2015 3:03 pm

Post Fri Jun 23, 2017 3:56 pm

Re: Unusual staining in 140-150 range

Hi Greg,
Thanks for your feedback. Yes I do have very high staining in some of the other channels above 150, even in 160+ range. I have a feeling it may well be eosinophils and will try out the Heparin treatment.
Interestingly I see more in normal BM than in AML.
Naeem
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GregBehbehani

Master

Posts: 80

Joined: Tue Apr 12, 2016 10:17 pm

Location: The Ohio State University, Columbus, Ohio

Post Fri Jun 23, 2017 6:33 pm

Re: Unusual staining in 140-150 range

I have seen that exact same phenomenon. The eos are present in the normal samples at a few percent (1-4% in my bone marrow samples) and very rare to non-existent in the AML samples.

If that's what you're seeing, the Heparin should fix it. Many thanks to Adeeb for figuring this out.

best,

Greg

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