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2016-Rahman et al-Cytometry A

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mleipold

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Post Thu Apr 14, 2016 3:29 pm

2016-Rahman et al-Cytometry A

"Heparin reduces nonspecific eosinophil staining artifacts in mass cytometry experiments"
Adeeb H. Rahman, Leticia Tordesillas and M. Cecilia Berin
Cytometry A, 2016
http://dx.doi.org/10.1002/cyto.a.22826

-note: this heparin block is specifically utilized in *intracellular* staining (ie, post-perm). Whole-blood was collected in heparin tubes, but presumably little remained after washes prior to perm.
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mleipold

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Post Thu Apr 14, 2016 3:32 pm

Re: 2016-Rahman et al-Cytometry A

Most labs I know collect whole-blood in heparin tubes.

I'm not aware of anyone doing a formal testing of EDTA vs citrate vs heparin vs (etc) blood collection tubes and any effects this may have on surface staining with CyTOF antibodies, but it could be interesting to see if the heparin would make a difference in WB staining there as well.
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GregBehbehani

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Post Thu Apr 14, 2016 6:52 pm

Re: 2016-Rahman et al-Cytometry A

Thanks for posting (and publishing) this!

I have seen a similar background population in human bone marrow, however, I primarily saw the background binding before intracellular staining, but after red cell lysis using the Smart Tube protocol (the heparin from the blood collection would have been washed away). The cells I saw were obviously not real as they were brightly positive for most everything in my panel, so I gated them out assuming they were debris. Interestingly, the cells were only about 1% of total events in normal bone marrow, but much lower (and often completely absent) in patients with AML. The experiment was barcoded so the abnormal events were most likely cells at some point to have been correctly barcoded. This is potentially consistent with an eosinophil population, or at least some other mature cell type.

I'll try your protocol on my samples and see if I can remove the background staining and, if I can clean it up, what normal population comes back after those cells are restored to normal staining.
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AdeebR

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Post Thu Apr 14, 2016 7:03 pm

Re: 2016-Rahman et al-Cytometry A

Hi Mike,

I don't think I've ever done a formal head to head comparison on the same sample, but we've successfully performed whole blood staining CyTOF using blood collected in EDTA, citrate and heparin tubes and haven't noticed any obvious differences.

The non-specific binding effect discussed in this paper was really only seen in blood post fixation (using both BD FACSlyse and SmartTube buffer) and exacerbated post permeabilization with saponin, triton X-100 or methanol.

The associated FCS files are publicly available here:

https://flowrepository.org/id/FR-FCM-ZZLL
https://flowrepository.org/id/FR-FCM-ZZLK

Adeeb
Adeeb Rahman
Icahn School of Medicine at Mount Sinai, NYC
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qinner

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Post Thu Oct 13, 2016 8:01 pm

Re: 2016-Rahman et al-Cytometry A

Recently we did Ab validation, and we found the same problem that the non-specific binding of Abs after permeabiliztion.
Has anyone fix the problem?
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mleipold

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Post Mon Oct 17, 2016 2:49 pm

Re: 2016-Rahman et al-Cytometry A

Hi Qin,

Could you be more specific about what sample type you were using, your exact stepwise staining procedure, and which antibodies were giving you the non-specific background?


Mike
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GregBehbehani

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Post Mon Oct 17, 2016 4:21 pm

Re: 2016-Rahman et al-Cytometry A

Hi Qin, Mike, and Adeeb,

The heparin treatment described in Rahman et al. (performed during antibody staining) almost completely eliminates this background staining in our hands. We performed an experiment similar to what was published and got very similar results. We also routinely collect in heparin, but this alone is not adequate, the heparin needs to be present at the time of antibody staining (or possibly just before).

I suspect the published protocol will work for you as well.

Good luck,

Greg

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