Issues with Lyosphere beads-background, especially CD8+
This is going to be a long post. I also assembled example data into a Powerpoint file, but it's too big to post (stupid 2MB attachment limit....).
Therefore, you can find the PPT (or, broken down by Trial at this Google Drive: https://drive.google.com/drive/folders/ ... sp=sharing
We had some Lyospheres made by Biolyph from my standard CyTOF PBMC surface phenotyping cocktail. A few labs at Stanford (Nolan and Blish, in particular) had been successful with this, and I think some other labs outside of Stanford have as well. I have used this cocktail for 6 years now, and it's been reliable.
For historical reasons, I still use the DN3 polymer from Fluidigm. I labeled 4 preps each of the 33 antibodies in the cocktail, which was theoretically enough for 450 1-test Lyospheres. We used a particular excipient that was basically TBS, and had been successful for the Blish Lab. I sent the antibodies as 33 individual stock tubes (since we and others have had issues with background if the antibodies are mixed too far in advance). Biolyph returned to us ~375 1-test Lyosphere.
I had retained a fair amount of the original Liquid stocks (aliquots of which I had sent to Biolyph), so we could do head-to-head comparisons of Liquid vs Lyo when we got the shipment back. I used a healthy control PBMC donor that we have characterized well over the years, and used in the titration of the Liquid reagents for the Lyosphere calculations.
Not unexpectedly, one antibody did not retain activity after being included in the Lyosphere. However, a later experiment demonstrated that it could be spiked in as a liquid reagent, so it's not a big deal.
However: I had much higher background of the Lyosphere cocktail (which I did put through the 0.1um spin filter as always, and as I did the Liquid cocktail made fresh) of certain antibodies on certain cell types. TCRgd was one. So was CD56, but it was weird: the background was worse on NKT (CD3+ CD56+) than on NK cells (CD3-, then CD56mid CD16+).
In the 2nd trial, I tried doing extra washes, and larger volume washes. No effect. CD8+ cells are most affected by background.
In the 2nd trial, I also used a second PBMC donor that we have characterized. Oddly enough, this donor did *not* have any of the same background issues as the first donor (CD56 on NKT was appropriate, etc).
In the 3rd trial, I tried diluting the antibody to 0.5x, as well as blocking with low-MW PEG or heparin (as Rahman et al Cytometry A 2016, though it was intracellular background, there). Again, one donor consistently affected, one donor consistently unaffected. Blocking consisted of incubating the cells with the block in PBS, then adding the antibody (still in the presence of block). CD8+ cells are most affected by background.
In the 4th trial, I tried blocking (same way as above), using BD Human Fc block, total mouse IgG, and total human IgG (based on Andersen et al Cytometry A 2016, which was focused on background on monocytes). Again, same donor affected, other donor still unaffected by background. CD8+ cells are most affected by background.
In the 5th trial, I tried testing more donors, as well as replicate draws we had of the affected donor. This was to test both processing conditions (ie, maybe something had been screwed up in the processing of the affected donor), as well as donor variability. In short: about half the donors were affected, about half the donors weren't. In one case, two donors were processed by the same person, same media, same process, on back to back days; one was affected, one wasn't. I couldn't find a gender correlation, or age. I don't have CMV data on all the donors, but at least one affected donor was CMV- (the first affected donor was CMV+).
I showed this data to some other people; one person mentioned a weird CD16 polymorphism, and suggested that I look at CD16 expression on CD8+ cells. That *did* correlate reasonably well, though again, at least one donor that was affected had at least a decent number of CD16+ CD8+ (though much fewer than most affected donors). But the IgG or Fc blocks made no effect (trial 4 tested).
In summary:
1. Some donors affected in Lyosphere sample, some not. All are fine in the corresponding Liquid sample (always run on the same day, fresh, as the Lyo trials; same liquid master stock as was sent for Lyosphere synthesis).
2. CD8+ affected more than any other cell type.
3. Blocking with heparin, LMW PEG, BD human Fc block, total human or mouse IgG made no difference.
4. Roughly 50% of the donors tested were affected. No obvious correlation with processor, processing method, date of processing, nor with CMV. Some correlation with CD8+ CD16+, but not perfect.
5. Unlikely to be DN3 issue, as Liquid reagents also DN3. Similarly, some donors fine with Lyosphere cocktail.
6. CCR7 (affected on CD8+) and CD85j (the antibody that lost activity) were from R&D Systems, purchased as lyophilized antibody. Therefore, technically, Lyosphere synthesis would be their second lyophilization. However, CCR7 fine on some donors, and generally fine on CD4+ even on affected donors.
6. Additionally, TCRgd was liquid stock from Biolegend, and was affected.
I want to be clear: I don't blame Biolyph for this. They had been successful with several other groups, so there was no way to know this would happen to us. They offered to work with us on figuring this out, but we already sank $25-30K into this, and aren't overly thrilled with the idea of spending more only on the chance that we find a method that will work.
For anyone thinking of Lyospheres (or any other lyo/dry method), I will suggest:
1. Start small: maybe 100ug of each, for 50-150 beads. Biolyph said that they can go as low as ~20 beads per synthesis. Only if those work should you go ahead with a larger prep.
2. Test *multiple* donors. In some ways, I got lucky (?): the first donor I tested was Affected, otherwise I wouldn't have known until I did my first plate of samples.
3. Examine every cell type you can think of. I have no idea why CCR7 is more affected on CD8+ than on CD4+.
If anyone has any ideas, we would greatly appreciate suggestions: as I said, we have $25-30K sunk into this, and would like to use the remaining ~300 Lyospheres sitting in our cold room...
Thanks,
Mike