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Does anyone experienced spillover or contamination like this

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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Mar 19, 2015 5:51 pm

Re: Does anyone experienced spillover or contamination like

Hi Komal,

Thanks for the pictures of your gating.

As I understand it, you do
1. Ungated-->Ir193 vs Event length to get Intact Singlets.
2. Intact Singlets-->CD45 vs Event length to get CD45+
3. CD45+--> CD4 vs CD8

Did you have CD66, CD14 or CD33, and CD3 in your CyTOF panel?

If so, I would advise revising your gating:
1. Ungated-->Ir193 vs Event length to get Intact Singlets.
2. Intact Singlets-->CD45 vs CD66 to get CD45+CD66-
3. CD45+CD66- ---> CD33 vs CD14 to get CD33-CD14-
4. CD33-CD14- ---> CD3 histogram or CD3 vs CD20 (or CD19) to get CD3+ (CD20-/CD19-)
5. CD3+ --> CD4 vs CD8

The reason for this is that it's possible that your CD3 depletion was not as successful as you would like. If so, then CD4+ and CD8+ cells would have made it through your purification.



Regarding MACS beads: while the beads themselves *are* iron oxide, iron is 54Fe-58Fe (92% 56Fe). Therefore, it won't be in the mass window of even a CyTOFv2, which is good, considering the amount of iron in your buffers, water, and cells.

In other words, it's the metal contaminants *in* the beads that are the issue, not the iron itself.


Mike
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komalkumaralienboy

Contributor

Posts: 44

Joined: Tue May 20, 2014 2:24 pm

Location: Linkoping University, Sweden and Finnish Institute of Molecular Medicine, Finalnd

Post Fri Mar 20, 2015 7:25 am

Re: Does anyone experienced spillover or contamination like

Hi Mike,
Thanks for the information.
I do not have CD66, CD14 and CD33 in the panel.
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri Mar 20, 2015 3:08 pm

Re: Does anyone experienced spillover or contamination like

Hi Komal,

Do you have CD3 in your panel? I know you used it in your depletion, but having it in your panel as well might allow you to check for instances where your depletion didn't work so well.

Granted, if most of the CD3 surface epitopes have been bound by CD3-biotin, you might not get much signal from your CD3-Ln. But it's at least worth a try.


Mike
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hwtchen

Participant

Posts: 9

Joined: Thu May 07, 2015 6:35 pm

Location: Sickkids, Toronto, Canada

Post Thu May 07, 2015 7:15 pm

Re: Does anyone experienced spillover or contamination like

Hi,
One of my users is considering pre-enriching their samples with Milytenyi automacs reagents. I am wondering if the contamination mentioned was only observed in positively selected population? Or anything that passes through the column would be exposed to this "some times' present contamination (this is what I would expect)? I am also curious as to which metal contaminants have been observed and at what level?
Tina Chen
CyTOF Research Project Coordinator, Guidos Lab
CyTOF Operator, Sickkids- UHN Flow Cytometry Facility
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu May 07, 2015 7:35 pm

Re: Does anyone experienced spillover or contamination like

Hi Tina,

We had a time or two where we got a Tin (Sn 112-124) contamination, when someone used MACS beads. But other times the same person used the same reagent (though a different vial/lot) and we were fine.

So, in our experience, it seems to vary a lot, from bead lot to lot, and also some evidence that it can vary between bead type or manufacturer.


The simplest thing to do would be to try to get a small aliquot of the beads, dissolve them in ultrapure HCl or HNO3, and run a high-dilution in liquid/tuning mode. I know a couple years ago, Fluidigm (then DVS) did some testing around this, so you might contact them and see if you can get their protocol/recommended procedure.


Mike
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elinastar

Participant

Posts: 19

Joined: Sun Nov 17, 2013 1:46 pm

Post Thu May 21, 2015 7:42 am

Re: Does anyone experienced spillover or contamination like

Hello everyone,

We have recently faced a major contamination of our runs in multiple channels, ranging from 139-148. This contamination originated from a new batch of BSA we obtained recently from Sigma. Since it was BSA, the false positive staining resembled an Ab staining by its appearance, but was non-specific and yielded weird populations. You might want to try processing your samples in PBS instead of CSM, you might find out that these double-positive populations are gone.

Happy CyTOFfing :)
Elina
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