Cadmium

[javells]

Dear All

I wanted to ask about using cadmium in mass cytometry. It is a toxic metal compared to what we are currently using. I having been using qdot which contains cadmium , but It never occurred to me how toxic cadmium could be. Trying to conjugate it to antibodies will probably increase the exposure risk. Similarly, osmium and ruthenium tetroxides are very toxic and were used in mass cytometry before with appropriate safety measures and all steps were carried out in fume hood.

- Has anyone tried enriched cadmium isotopes and do they work with with maxpar conjugation kits ?
- What would you think and suggest regarding using this kind of hazardous metals in mass cytometry ?

Thanks

Muharrem

[ChristophS]

Hi Muharrem,

QDots can be used on a CyTOF but I have not found them very attractice exactly for the reason that they "burn" a range of channels (natural abundance Cd and natural abundance Te). I have tested natural abundance Cadmium and Tellurium for chelation in X8 and both DONT work. For the toxicity: if you have been chewing/licking vour colorful wooden toys as a toddler (before the 1970s) you probably ingested more Cd than by drinking a vial of Qdots .

Hope this helps.

Best

Christoph

[mleipold]

Hi Muharrem,

Qdots have been passivated with surface coatings that reduce their toxicity. While formally they do get atomized and ionized ("burned completely") in the CyTOF plasma, I don't think they're much more of an issue in the exhaust than any of the other metals we use.

Regarding enriched Cd salts: I know that Henrik Mei tried loading Cd2+ into the ITCB-EDTA chelator, but it didn't work as well as Pd2+. He only tried 1-2 times, so it's unclear whether it was a chemistry problem (pH, solubility, oxidation state changes, etc), or whether it fundamentally just didn't want to go into the EDTA chelator and *remain* there stably.

Maybe he'll chime in, as he might remember more of the trials....


Mike

[ErinSimonds]

Cadmium is primarily a +2 ion (sometimes +1), so it won't work with the Maxpar chelator which requires +3 ions. It is possible to chelate cadmium with EDTA, as MIke suggested. Infusing humans with EDTA causes cadmium excretion in the urine (ref).

On the potential exposure to toxic levels of cadmium: I was surprised to learn that one serving (46g) of sunflower seeds contains 23.9 ug (212 nanomoles) of cadmium (ref). The concentrated metal stocks we use for Maxpar labeling are 50 mM. If I did the math correctly, one serving of sunflower seeds contains the same amount of cadmium as 4.3 uL of a 50 mM cadmium stock. Admittedly, the cadmium in sunflower seeds could be much less bioavailable than a cadmium salt solution, but I just want to make the point that we're not handling very high levels of metal in a typical CyTOF workflow.

[javells]

Thank you all for suggestions and advice !

It seems that DTPA wouldn't work for +2 ions, as cadmium.

I was thinking whether Isothiocyanobenzyl-EDTA would work as a chelator for CdCl2 as it works for Palladiums (+2) and palladiums are also 2+ ions.

Mike, Is there a reason for Henrik Mei choosing ITCB-EDTA over Isothiocyanobenzyl-EDTA as a chelator?

[mleipold]

ITCB-EDTA is the same as IsoThioCyanoBenzyl-EDTA.....just faster (and less of a mouthful when speaking it).

Like I said: I don't know that it *can't* work. Just that it didn't work in the 1-2 times Henrik tried it.....we didn't chase this very hard, so it's entirely possible it was something as simple as a pH issue, rather than something more problematic like an oxidation state change that might make it fall out of the chelator.

[javells]

Right , my bad !
I'll let you all know if it works

[ssivajothi]

Hi Muharrem,

Did you end up trying the Cd conjugation with ITCB-EDTA? Interested in hearing your experience.

Thanks,
Santhosh

[mleipold]

Hi all,

With the Cadmium MCP9 polymer kits now being sold by Fluidigm, I have done some tests. Our initial plan is to use them in live-cell antibody-based barcoding (CD45, etc); easier than doing the ITCB-EDTA conjugations from Henrik's papers (though his new protocol has significant improvements).

Fluidigm sells 7 kits: 106, 110, 111, 112, 113, 114, 116. There is also a 108 isotope in nature, but presumably it is not available in sufficient purity to sell. I purchased 6 of the 7 kits: I did not purchase 113, as we already use In113 for antibody labeling with X8/DN3 (I didn't want any of my coworkers to mess up an experiment by putting both an antibody and a barcode antibody on the same channel).

The labeling procedure is roughly similar to X8/DN3, mainly just longer incubation times in certain steps. When I received my kits, they didn't come with protocol sheets; my understanding is that has now been fixed, but contact your FAS (or try to find it on Fluidigm's website) if you don't get a copy with your order.


In short: they work well for live-cell barcoding on PBMCs. I only tried one concentration (3ug/mL) for the singles (1 of 6) and trios (3 of 6), but the positives for 3-of-6 were just above 1e2, the negatives were basically below 1e1, and essentially baseline resolution between the pos and neg populations. For single Cd staining, the positives were almost at 1e3.

The 11/22/19 PDFs are from single Cd CD45, no combining into a barcode sample. That shows the spillovers into the other Cd channels.

The 11/27/19 PDFs are from a 3-of-6 barcode experiment, with 3 separate healthy donors (including a biologically CD33neg donor, who is great for debarcoding efficiency checks).



All were at 3 ug/mL; we will test going lower, as well as a few more barcode combinations. Assuming they turn out, then we can test freezing them. 3/6 would give the "standard" 20 barcodes. If you started including Fluidigm's 89Y-CD45 or the 113Cd kit, that would give you 7 channels, for 21 (2/7) or 35 (3/7) barcodes.

But we're going ahead with labeling more CD45 for some other projects. Right at this moment, we don't have immediate plans to test other markers on the cadmiums. I would make the point that the data that Thiru (I think) showed at the June UGM in Vancouver wasn't hugely impressive to me for *every* surface marker moved down onto the Cd channels: CD8 looked fine, but CD4 or CD16 was less optimal, in my opinion. But I hope that this info about CD45 signal intensity gives people a place to start for their own panel designs.


Now let me speak directly to Fluidigm: THIS IS A GREAT OPPORTUNITY TO OFFER PRECONJUGATED Cd-CD45 ANTIBODIES FOR LIVE CELL BARCODING. PLEASE MAKE THESE AVAILABLE IN YOUR CATALOG......THIS IS LOW-HANGING FRUIT THAT A LOT OF PEOPLE WANT.


Mike

[Jahangir]

Hi All,

Thanks Mike for your thorough testing. This message might be slightly off topic - apologies. You mentioned you can use additional channels to the Cadmiums to expand the barcoding complex to a 7-choose-3 combination.

My question is, why a 7-c-3 over a 7-c-4? Or is there any benefit from one over the other? Wouldn't the latter create a greater redundancy in the barcoding system? So you would get less cells which are assigned to the wrong channel? Whilst still retaining the highest combinations possible. Similar to a 9-c-4 or a 9-c-5.

Many thanks,

Jahangir