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Monoisotopic Cisplatin 198Pt spill

PostPosted: Tue Jan 11, 2022 2:05 pm
by taylorwitte

We were looking at switching to monoisotopic cisplatin from natural abundance to open up some channels in our panel. We titrated the monoisotopic cisplatin on 3 million reference + PBMC cells, half of which were heat killed. The old sample in our titration is natural abundance cisplatin at our working dilution. We are seeing a ton of spill form our monoisotopic cisplatin. Has anyone had any luck minimizing the spill from monoisotopic cisplatin so they can use the other platinum channels?

Re: Monoisotopic Cisplatin 198Pt spill

PostPosted: Wed Jan 12, 2022 9:43 pm
by mleipold
Hi Taylor,

I think there are a few things going on here.

1. You can afford to go even lower on your cisplatin titer than 1:8000, as your positive population is still above 1e2.

2. You're always going to have *some* 194/195/196 spill in your 198Pt stock (whether cisplatin or the new CD45-Pt Fluidigm sells, which they make from the cisplatin stocks). This is true for all the "monoisotopic" cisplatins. In fact, "monoisotopic" isn't really technically correct: "isotopically-enriched" is more accurate.

The issue is probably worse of 198Pt because it's lower abundance in nature (7.16%) than 194Pt (32.97%), 195Pt (33.83%), or 196Pt (25.24%). Remember, it's easier (and cheaper!) to get rid of a minor impurity. For example, it's fairly easy for Trace Sciences (or anyone else) to enrich natural abundance 115In from 95.71% to 99+%. It's harder to enrich natural abundance 113In from 4.29% to ~93% (with 7% 115In impurity still there), and it would be really expensive to go up to 99+%. Similarly, this is one reason why enriched 102Pd 1.02% nat abund) is 10x the price of any of the other enriched Pds.

So, your 198Pt stock is going to have a higher 196Pt spill than your 196Pt stock is going to have as a 198Pt spill.

3. Consider *how* you're using the different Pt channels in your experiment. For example, your 198Pt-cisplatin: yes, you *do* have 194/195/196 spills, but that's only in the 198Pt-bright population, which are your *dead* cells. You're going to be gating the dead cells out anyway, so you're going to be getting rid of those specific spills from the 198Pt-cisplatin.

Even if you're using the 194/195/196 channels for live-cell barcoding, I don't think there would be a problem. First, the BC channels would probably be above 1e2 for positive cells, while your spills from 198Pt cisplatin into the BC channels remain a bit below 1e2 . That's probably good enough for the algorithm to separate them, especially if you include 198Pt=0 in the debarcoding key. Second, even if the Livedead spills would interfere, that would code as an extra BC channel, which would violate the debarcoding key and get tossed out. Since that would only apply for your Dead cells, the algorithm would basically be throwing out some of your dead cells.