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Palladium barcode artifact when staining PBCs (not PBMCs)

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jcvillasboas

Contributor

Posts: 41

Joined: Fri Apr 03, 2015 3:22 pm

Location: Rochester - MN

Post Thu Jun 18, 2020 10:07 pm

Palladium barcode artifact when staining PBCs (not PBMCs)

We were recently testing the difference in staining patterns on peripheral blood cells (PBCs) in comparison with peripheral blood mononuclear cells (PBMCs). PBCs were created simply by spinning whole blood and separating the buffy coat from serum/plasma. PBMCs were created through standard gradient density separation using Ficoll. Both were stained fresh with our standard panel in separate tubes. We included another tube with our cryopreserved PBMC reference (Ref1). All tubes started out with 3 x 10^6 cells. During overnight intercalation they were barcoded with the Pd-based barcoding kit from FDG as we normally do. They were run separately and the only reason I wanted to barcode them was to see if there was a differential uptake of barcodes between mononuclear cells, residual red cells, granulocytes, and debris.

When I inspect the files using [Pd channels] vs time it becomes clear that the PBC tube has very low barcode signal on the expected channels (104/106/108). This is not seen on the other tubes:

Capture.PNG
[Pd channels] vs time


Although possible, it is very unlikely we failed to add adequate amount of barcode to the PBC tube. Besides, when I inspect the signal on the iridium channels and on CD45 I see that the Ir signal is very low and there is a sizable populations of CD45low cells which could represent RBCs (PBC sample did not undergo red cell lysis step):

Capture1.PNG
Inspecting Ir channels and CD45


I hypothesize that the red cells in the PBC tube are causing interference and quenching both the Ir intercalator and barcode. Not sure how that could be possible as far as the iridium intercalator goes since mature RBCs are not supposed to have much nucleic acid left. I am puzzled and would like to know if anyone else has seen this phenomenon. Any thoughts or comments are welcomed.

Best

JC
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GregBehbehani

Master

Posts: 80

Joined: Tue Apr 12, 2016 10:17 pm

Location: The Ohio State University, Columbus, Ohio

Post Thu Jun 18, 2020 10:53 pm

Re: Palladium barcode artifact when staining PBCs (not PBMCs

Hi J.C.,

Your hypothesis is correct.

Both the intercalator and the the barcoding reagent bind to protein. (In spite of what its name implies, the intercalator is not particularly specific for nucleic acids; I have confirmed this in a variety of ways.) Both will stain in a relatively direct correlation to the total protein in the tube (this is why both have to be stained in PBS without any BSA or other protein). Typically, you can just stain based on cell numbers (which is what we recommended in the papers on the barcoding protocols), but if you had contaminating red cells (which I would assume you wouldn't have counted as cells) they will suck up both intercalator and barcode. Platelets will also stain with both and will use up the reagents (though in practice, there aren't enough platelets to cause an issue). In your case, either red cells (or red cell fragments) probably bound up the barcode and the intercalator. In general, red cells will not survive a typical CyTOF staining protocol, so they would be gone by the time you ran your sample, but they probably were around long enough to suck up your reagents.

We use PBCs as a control most of the time; it's fine to do this, but you always lyse the red cells first.

best,

Greg

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