Barcoding combined with Maxpar Direct Immune Profiling Assay

[DanielBachurski]

Dear all,

Our Lab recently purchased the MDIPA kit to analyze PBMCs from leukemia patients. For this clinical setting, a barcoding approach fulfilling the following bullet points would be ideal:

- Does not require fixation & (transient) permeabilization before barcoding so that the surface stainings are not altered
- Does not interfere with Intercalator 103Rh as its part of the MDIPA kit and cannot be removed from the panel
- Allows for barcoding between 10 - 20 patients samples
- Does not cost 1,000,000 USD or requires a Ph.D. in inorganic chemistry

From my understanding following channels could be used for such a barcoding approach (102Cd&Cd104 (not sure about the interference with 103Rh though), 106Cd,110Cd, 111Cd, 112Cd, 113Cd, 114Cd, 116Cd.

142Nd, 159Tb, 162Dy, 165Ho, 169Tm, 175Lu, 209Bi are all used up for additional markers.

Any help or hint would be highly appreciated! Also, your experience with similar barcoding requirements for your panels would help a lot.

Thank you all so much!

Best wishes from Cologne
Daniel

[mleipold]

Hi Daniel,

A couple comments:
1. There are no 102 or 104 isotopes of CD. I think you're mixing them up with the Pd 102 and 104 from the Fluidigm BC kit.

2. To use the Fluidigm BC kit or the Bodenmiller and Zunder small molecule BCs, you have to perm the cells at least transiently. Therefore, you have to fix the cells prior to BC and perm.

The main non-perm BC option is to use an antibody based BC like CD45, CD298, and/or b2m.

3. Pd102 and Pd104 don't really spill into Rh103, not vice versa. There is no Pd103, nor Rh102 or R&104.

4. For the live-cell antibody based BC, the Cd channels work well.
viewtopic.php?f=7&t=880&p=4621&hilit=Cd+kits#p4621

[markUofT]

Hi Daniel,
We might be able to help you out for less than 1 million dollars. See below.
Send me an email and we can connect.
Best,
Mark

https://sites.chem.utoronto.ca/nitz/con ... ixed-cells

[DanielBachurski]

Hi Mike,

Thank you for your detailed response! Your help is much appreciated!

1. You are completely right! I was mixing up Cd with Pd for the isotopes 102&104! Sorry for the mistake!

2. In your experience or to your knowledge, does it work well to transiently permeabilize PBMCs with 0.02% saponin without major interference with the surface staining, or would you rather recommend the antibody-based bc which could be more difficult to establish? What is the most common approach here?

3. Great, so this opens up the possibility to use the bc kit from Fluidigm! I was told beforehand that there might be issues combining Rh103 and Pd102&104.

4. If anyone is interested to share or sell antibody-based bc (excluding CD45), please let me know!

Best regards
Daniel

[mleipold]

Hi Daniel,

You're going to have to figure this our for your system, unfortunately.

For example, if you want to use CD45 as a staining marker, then you'll have to make a decision.
1. Use CD298 and/or b2m as the BC and use CD45 only as a surface marker (ie, not as a BC)
2. Use CD45 as a BC, and make the assumption that anything that gets through the BC debarcoding is inherently CD45+. This is generally a good assumption, but you could miss some CD45lo.
3. Use CD45 as a BC, but then use a separate clone for CD45 as a surface marker.

Stanford HIMC has run into some issues with certain markers. For example, one project we're working on, we're having some difficulty getting CD45, CD298, or CD45+CD298 to work on 100% of CD34+ cells. We're getting about 70% coded, but that means we still lose about 30% of CD34+ during debarcoding. We haven't chased this exhaustively, as that customer decided to halt development and go back to the Fluidigm BCs.


Transient perm is going to be better than a permanent perm like Methanol, but it's still a perm, and still requires fixation ahead of time. So, you're going to have to test whether your drop-ins are going to function sufficiently for you to have resolution in your gating.


Regarding Mark's Te-barcodes: frankly, I've been pushing Fluidigm to release them since Mark published his paper, since they work on both live *and* fixed cells.

Then again, I've also explicitly called out Fluidigm for over a year to release Cd-labeled CD45, CD298, and b2m for live-cell antibody-based barcoding (and actually, since the antibody-barcoding was first published 5yr ago or so).

So, push your FASs on this.


Mike

[DanielBachurski]

Thank you, Mike, and thank you, Mark!

Mark's Te-barcodes seem like the best way to label the patient samples for my purpose! I am happy to discuss it with him! As you indicated, CD45 is already included in the MDIPA kit and thus not the ideal choice for an antibody-based barcoding approach.

I completely agree that there should be more commercially available barcoding options, that ideally do not require fixation (permeabilization) of cells.

Happy to learn more about mass cytometry experiments from you all!

Best wishes
Daniel