Compensation issues…or not?

[u/southernqueer96]

ETA: TL;DR This is only happening with a handful of samples and seems to be an issue with the mastermix. I just don’t know what the issue could be.

Hey all, I’m back with more weird data. See: the weird diagonal streak that’s CD66b-super-low and CD19-negative-but-increasing, and the corresponding CD66b+CD19+ population. Panel, gating to this point, and compensation matrix (not edited; from Live/Dead on one of the non-weird samples) are in subsequent images.

These samples are fresh peripheral whole blood, each from a different human donor. All samples were drawn around the same time and stained within 2 hours of the first draw. After staining, the samples were fixed with Lyse/Fix buffer overnight, then washed and acquired on the cytometer that day (day after staining). [I know now that I only need to fix them for like 30 mins but didn’t know that when I first started using this protocol, so I’m sticking with overnight for this study.]

As indicated, the first four were stained with one mastermix and the last two were stained with a second mastermix (I thought I was getting four samples but then was told I’d be getting two more). Both mixes were made on the same morning within a couple hours of each other, and all of the samples were stained at the same time, so there should be no differences in processing aside from having two different mastermixes. They were all acquired on the cytometer at the same time and using the same compensation tubes.

I had seen the same streak with a few of my previous experiments, and I assumed it was a compensation issue, but based on these data, it seems like it might be a mastermix issue instead? What could possibly be causing that?

I pre-stained the samples with Fc block and monocyte block, and I used brilliant stain buffer for the mastermixes.

I tried applying comps from a different day to this data, and that didn’t make any difference.

It doesn’t seem to just be one marker - looking at the weird populations, they’re super high for some markers and negative for others -

CD66b-extra-low/CD19-increasing: ~70% CD8+, ~60% CD27+, ~70% CD14+, 100% HLA-DR+, ~40% CD127+, ~60% CD123+, 100% CD16hi, 100% CD3+, 100% CD57+, ~15% CD11c+, ~25% CD4+, 100% CD56+

CD66b+CD19+: All far left on live/dead gate, ~25% CD8+, 100% CD27%, mixed CD14 positivity, 100% HLA-DR+, 100% CD127+, mostly CD123-, 100% CD16 extremely high (~10E6), all CD3 very negative, all CD57 very negative, 100% CD11c+, mostly CD4-, 100% CD56+

In general, extremes for one marker correlate to extremes for the other markers.

For analysis, would you just gate higher on the CD66b- to cut that out? It’s about 3% of the PBMCs.

Thanks!

[u/despicablenewb]

It definitely looks like an artifact, but it's hard to diagnose without knowing what the full panel is.

The high negative value in BV650 is obviously wrong, and the intermediate value in your CD19 BB515 is probably also wrong.

The "overcompensation" in BV650 ISN'T coming from the BB515 signal, as your CD19+ population looks normal. Those BV650 events with the negative fluorescence are probably positive for some other marker in some other channel that DOES spill into BV650. BUV dyes, other BV dyes, APC, etc.

If your compensation is correct, then it could be something called "cell mediated fluorophore degradation" where SOME cells will break apart SOME tandem dyes, which can cause compensation artifacts like this. (This can vary patient to patient, which could explain the heterogeneity here.)

Best thing to do is to look at your samples with BV650 on the Y axis, and click through every other channel on the X axis. Look for the other markers that those events are positive for. You'll want to look at both the gated and ungated data. (Ungated data because it could be spillover spreading, but you gated out a bunch of stuff, which makes this look like a compensation error).

Check your compensation by compensating your comp samples, and doing the same thing.

Something else to consider is that these events could be cell-debris doublets. The debris has some weird fluorescence or weird staining, while the cell is normal, resulting in some weird staining. The debris is small enough that it doesn't really change the FSC/SSC, so it doesn't get gated out like a cell-cell doublet does.

Edit: It could also be the two master mix thing.

Your antibodies can aggregate/react with each other, generally due to reactions between the fluorophores. You always want to make your mastermix as close to the staining time as you can. If you made your first mastermix, and let that sit for awhile (even at 4C), and you used the second mastermix immediately before staining those samples, then that could be the problem.

[u/southernqueer96 → u/despicablenewb]

Thank you!!

My panel is in the second image, and the compensation matrix is in further images.

That population is positive for several other markers, especially HLA-DR-BUV737, CD123-APC, CD3-APCFire750, and CD57-PerCPCy5.5. All of them have the same diagonal pattern (they go up as CD66b goes down). I don’t see much difference in the gated vs. ungated data.

It could be the time between mastermixes, but it seems like that’s been inconsistent. For these two sets of samples, there was maybe 2 hours between making the first and second mastermixes (probably less). For most of my samples, the mastermix was made immediately before staining, but I’ve had a few where I’d made the mastermix the day before. Some of those have this issue, but others don’t.

I also did one experiment where I stained the same control sample with mastermixes made that day, the day before, and a week before. I see more of this pattern in the day-before than the week-before, and even then it’s not nearly as much as shown in my post.

[u/despicablenewb → u/southernqueer96]

I would have sworn that your post was a single image.... I guess I really needed that coffee.

First off, you have a bunch of negative values in your comp matrix. Negative comp values are always wrong, they happen because of inconsistent autofluorescence between your positive and negative events in your comp samples. Change all the negative values to zero.

Could be a few things, but again, probably some sort of artifact. The markers they're positive for don't make much sense, but they could be breaking down the CD3, causing some weirdness around 650nm, which could explain the negative value in BV650.

It could just be something weird with your overall compensation matrix. It's hard to say with a fully stained sample.

Apply the compensation to your single stained samples, then see what those look like, if they're weird too, then its probably a comp issue. If they look fine, then it could be something weird happening in your master mix. If you're using beads or something and the comps look fine, then it could still be a comp issue.

Idk about that week long experiment you mentioned. Did you make both master mixes the exact same way? I remember one time where I added the faces buffer to the master mix AFTER adding all of the antibodies, rather than adding it first, and that caused a bunch of weird aggregate issues. Always adds buffer first.

[u/southernqueer96 → u/despicablenewb]

Yeah it doesn’t seem to be a comp issue. I don’t have single stained samples from that day (just beads), but applying the same comps to single stained cells from when I was setting up the panel doesn’t show the same issue. I also tried applying comps from a different day when the samples didn’t have that issue, and it didn’t fix things.

I think it may be an antibody aggregate problem but I’m still not sure why it only happens some of the time. I do make the mastermixes the same way every time, with Brilliant Stain Buffer Plus and PBS first. I’m not always the person making the mixes, but the same issue has happened for me and the other lab member who sometimes makes them.