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Cd metal and fresh whole blood staining issue

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oAMIRo

Participant

Posts: 4

Joined: Mon Sep 08, 2014 7:30 am

Post Fri Nov 18, 2022 4:26 pm

Cd metal and fresh whole blood staining issue

Hi All,

We are losing the Cd metal stain when staining fresh whole blood.
protocol is straightforward comparing 3 staining protocols.
A fresh whole blood sample split into 3 aliqutes:
1. WB--> smartube fix--> freeze -80--> thaw and lyse (smarttube reagent)--> heparin/Fc block--> extra stain--> BD fix/perm--> intra stain--> fix&Ir--> Helios.
2. WB--> Fc block--> extra stain-->Biolegend RBC lysis--> BD fix/perm--> intra stain--> fix&Ir--> Helios.
3. WB-->Fc block--> extra stain--> smartube fix--> freeze -80--> thaw and lyse (smarttube reagent)--> BD fix/perm--> intra stain--> fix&Ir--> Helios.
We use CAS plus with cell acquisition.
In the attached pdf live intact cells are plotted.

The Cd stain is normal with ST (1) but lost when we are staining prior to fixation (2, 3).
Any one else encountered this?
I'd appreciate any thoughts on this?
Thanks
Attachments
Amirs illustration.pdf
(85.07 KiB) Downloaded 119 times
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mleipold

Guru

Posts: 5450

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri Nov 18, 2022 5:32 pm

Re: Cd metal and fresh whole blood staining issue

Hi Amir,

Unfortunately, I haven't done staining with Cd/MCP9 in complete WB, so I can't give much direct insight.

Does this happen with every donor you try? I'd be surprised if serum/plasma were an issue here, but there *is* precendence for donor-specific effects with CyTOF antibodies (see initial MDIPA+WB issue here: cytoforum.stanford.edu/viewtopic.php?f=10&t=2211 )

I guess one thing to check would be what happens if you do a mock experiment using PBMCs. If you put PBMCs through each of your workflows, you'd at least be able to track what happens with 3 of your 4 Cd-labeled antibodies (all but CD66b). The Sinai paper demonstrated that the same donor-effects were absent when PBMCs were used; therefore, if you still see an effect, it would be more likely to be due to one or more of your steps/reagents than some weird donor biology.


Mike
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CRStevens

Master

Posts: 57

Joined: Thu Jul 17, 2014 5:07 pm

Post Fri Nov 18, 2022 6:31 pm

Re: Cd metal and fresh whole blood staining issue

Hi Amir,

We had this problem as well. When using conventional WB lysis reagents, we were losing Cd labels. It turns out Standard Biotools recommends using CalLyse buffer (this is what they used to test their whole blood protocol and had no clue BD or Lyse/fix effected Cd). I've just started using it for all my whole blood procedures. I actually think the procedure is easier and have noticed more consistent lysing. Hope this helps.

https://www.thermofisher.com/order/cata ... uct/GAS010
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oAMIRo

Participant

Posts: 4

Joined: Mon Sep 08, 2014 7:30 am

Post Fri Nov 18, 2022 7:53 pm

Re: Cd metal and fresh whole blood staining issue

Thanks Mike,
I did something similar with PBMCs already. Cd stain just fine.
In the attached file, PBMCs were stained like in workflow#2 omitting the RBC lysis and intra stain steps. The WB was stained as in workflow#1, again without the intra steps.
I will say that we routinely stain PBMCs intra and with Cd channels with no issues, so I hope this is not the missing part.

Hi Chad,
Indeed, my initial thought was it is something to do with the lysis buffer and this is in part why workflow#3 is there. but when it happened with two different buffers; one being Biolegend RBC and other being smartube reagent, I got puzzled.
I didn't expect it to happen with the ST one.

Thanks For the CalLyse buffer tip. Do you use it before or after the extra stain? Any effect on the more fix sensitive markers?
I ask because I see in the data sheet that it contains PFA, so cells are fixed while lysed.

Thanks a bunch
Amir
Attachments
Cd channels.pdf
(59.33 KiB) Downloaded 98 times
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mleipold

Guru

Posts: 5450

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Fri Nov 18, 2022 7:58 pm

Re: Cd metal and fresh whole blood staining issue

Hi Amir,

Especially since Chad has that new info about the lysis buffer issue, I would recommend a fully PBMC-mock experiment. *Including* the Lysis buffers, especially since that appears to be where the problem is occurring.

Just so you would have internal data demonstrating where the actual issue is (for future reference, both yourself and other lab members).


Mike
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CRStevens

Master

Posts: 57

Joined: Thu Jul 17, 2014 5:07 pm

Post Mon Nov 21, 2022 3:36 pm

Re: Cd metal and fresh whole blood staining issue

Hi Amir,

We routinely will get fresh whole blood, to surface stain, lyse/fix, then do with or without intracellular. When you stain on the fresh whole blood you dont have to worry about the PFA effecting your antigens. If your workflow requires that you use stabilized samples, then you will have to validate your panel accordingly. We've done Cytochex tubes for instance and had issues with CD45RA, CD8 and other i can't remember. It is really dependent on your clones and the stabilizer/fixation reagent.

As a side not for a longitudinal study if you can stain fresh and then freeze your sample after fixation it might be a better way to run it. We do it this way, then thaw all our samples all at once at the end of the study for Ir intercalation and acquisition. This allows for all your samples being run at once.

Anyway... the Cd is definitely an issue with buffers whether it is the lyse/fix or something else. They dont know what component it was, but we would see greatly diminished signal and sometimes even completely missing.

-Chad

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