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Tubes to use for blood collection

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Wkennedy

Participant

Posts: 5

Joined: Fri Dec 11, 2015 10:23 pm

Post Tue Jul 12, 2016 7:41 pm

Tubes to use for blood collection

Hi everyone,

We are looking to optimize our workflow for pheripheral blood sample analysis and are hoping to collect plasma, PBMC's and granulocytes from these samples.
For this, I was wondering if there happens to be a favoured tube amongst CyTOFers. When looking through the literature it seems that both EDTA and Heparin are used,
and when looking at other downstream applications it was mentioned that EDTA Isn't recommended for PCR because of the chelation properties. Would this follow a similar train of thought for the metal based analysis we are looking at in CyTOF (ie. use heparin instead of EDTA).

Looking forward to your feedback.

William Kennedy
University of British Columbia
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Tue Jul 12, 2016 9:34 pm

Re: Tubes to use for blood collection

Hi William,

I think there are a couple topics here.

1. I haven't seen any evidence that EDTA will rip lanthanide metal ions out of a MAXPAR polymer. In fact, my antibody staining buffer contains 2mM EDTA.

It's not easy for another chelator to pull away a metal ion already chelated, without some kind of denaturant or heat. Even then, it usually takes a stronger chelator (for that metal ion) and/or substantially higher concentrations of the second chelator to ensure that the metal ion doesn't go back in. Dojindo has a nice table of chelator stability constants (attached to this post). If you look at, say, Eu (III): EDTA has a constant of 17.35 while DTPA (in the MAXPAR polymer) has a constant of 22.39. Therefore, it would be difficult for EDTA to rip Eu(III) out of DTPA.


2. Removal of metal ions affecting antibody binding. This can be due to their recognition epitopes requiring a structural metal ion.

For example: DOI: 10.1002/cyto.a.20084
"Calcium chelators caused a dose-dependent decrease in fluorescence intensity, using specific anti-human CD8α mAbs. This phenomenon was not due to CD8 internalization and could be reversed by the addition of calcium ions. In contrast, calcium depletion increased staining intensity with one anti-CD8β mAb."


So, I think ultimately, you're just going to have to test which is better for your purposes and your panel(s). The vast majority of the PBMCs we run are from heparin tubes.


Mike
Attachments
Chelate_Table_of_Stability_Constants.pdf
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Wkennedy

Participant

Posts: 5

Joined: Fri Dec 11, 2015 10:23 pm

Post Wed Jul 13, 2016 3:16 am

Re: Tubes to use for blood collection

Perfect! Thanks for the help. Do you think this logic would extend to the barcoding reagents as well?

I would think that if the antibodies were safe it would make sense that the barcodes are as well? Or do you think that rather than taking away the palladium the EDTA could actually replace the barcode in the cells in question (therefore having free EDTA and no barcodes in the cell).

Thanks again,

William
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed Jul 13, 2016 3:04 pm

Re: Tubes to use for blood collection

Henrik showed in the first Pd-CD45 barcoding paper that different chelators couldn't rip the Pd out of the EDTA once it was loaded in, and that attached to the antibody. So, I wouldn't worry about that too much.

Fluidigm won't state what their barcoding agent is, aside from that it contains Pd and that it cannot be used to label an antibody like CD45. That said, I don't think EDTA would interfere with their barcoding agents, as EDTA doesn't covalently react with really anything inside a cell. For the same reason, I don't see free EDTA preventing the SCN-EDTA-Pd barcoding agents from Zunder et al from working: free EDTA shouldn't compete with the SCN for cellular amines.


Mike

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