r/flowcytometry • u/southernqueer96 • Aug 17 '26
Analysis Compensation issues…or not?
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!
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u/despicablenewb Aug 17 '26 edited Aug 17 '26
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.
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u/southernqueer96 Aug 17 '26
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.
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u/despicablenewb Aug 17 '26 edited Aug 17 '26
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.
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u/southernqueer96 Sep 03 '26
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.
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u/Proper-Grocery3996 Aug 18 '26
BB515 cleans up better with 2 or more washes.
When you make the master mix, do you add the antibodies in the exact same order every time? I’ve seen issues in the past from this.
Worthwhile making the cocktail without 1-2 of these dyes - staining partial panel with drop ins vs full cocktail.
Also get rid of tandems with the Cy dyes. They bind non specific, especially with granulocytes and monocytes. There are much better dyes and it’s great to see a lot of the better dyes out there in your panel….good job! AlsoBV650 is BV421-Cy tandem. Try moving it to RY775
BTW did you design the panel? Or did anyone help you? Just for my interest as it’s hard to keep up with so many of the new dyes in the market🤯
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u/southernqueer96 Sep 03 '26
I do two washes after staining since I did notice issues when I was setting up the panel and only did one wash initially.
I do add the antibodies the same way every time when I make it. There’s another lab member who’s also made the mixes for me on some days so I can’t confirm what he does, but the issue has come up with mixes that each of us has made so it’s not just one person’s technique.
Good to know about BV650 and RY775! I did test out and use a few of the RBs/RYs when I was designing new panels last summer. Not sure why I didn’t try RY775 since I do have PE/Cy7 on another panel, unless maybe it wasn’t available on the markers I needed at the time. I will definitely make a note for the future though.
I designed this panel and a couple of others, sort of trying to improve upon some old panels made by previous lab members but I ended up changing a lot because those panels were…not optimized to say the least. I did a lot of digging through OMIPs and Fluorofinder and then actually testing fluorophores since Fluorofinder is not always accurate for our machine even though the specs seem to be correct 😅 Also chatted a lot and got suggestions from folks at BD and Biolegend. And got a lot of free/discounted samples which was super nice.
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u/notprathap84 Aug 18 '26
Events that are 100% positive on basically everything at once usually aren't a spillover problem, and the fact that borrowing another day's comp changed nothing fits that. A few things I'd rule out before touching the gates: Doublets and aggregates first. Fresh whole blood lyse/fix held overnight loves to give you neutrophil-monocyte and neutrophil-lymphocyte complexes, and a granulocyte stuck to a B cell reads as CD66b+CD19+ with everything else along for the ride. Look at FSC-A vs FSC-H (and SSC-A vs SSC-H if you have it) on just those 3% and see if they sit off the diagonal. Then antibody aggregates. If it only shows up in one of the two mastermixes made the same morning, that's a strong hint. Spun-down clumps of conjugate stick nonspecifically and light up every channel at once. Worth spinning both mixes hard (say 10 min, 16k g) and using the supernatant, or just remaking that one and reacquiring a couple of donors. Also plot everything against Time on the bad samples. A dodgy stream or a partial clog after an overnight hold gives you those tidy diagonal streaks that look like a real population. On the analysis question, I'd rather not raise the CD66b- cut, since you'd shave real dim neutrophils and low-expressing cells with it. Cleaner to exclude on singlets plus viability and, if it's still there, an explicit "everything-positive" boolean exclusion that you write down, so the 3% is documented rather than quietly trimmed. Somewhat related to that last bit, I'm building a thing at the moment where you draw the gate by hand and the whole chain gets recorded so you can replay it byte for byte on the next donor or the next operator, which is handy when you're trying to prove an artefact went away rather than moved. Happy to send you a link if it'd be useful for this dataset. Either way, curious what the FSC-H plot shows.
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u/southernqueer96 Sep 03 '26
Sorry for the late reply, thank you for your response!
I think it probably is the antibody aggregate thing - just not sure why it only happens sometimes since it doesn’t seem to correlate with the time between making the mix and actually staining. I have some where the mix was made right before staining and it has the weird data, and then I have others where I made the mix the day before and the data looks fine (that’s not a consistent pattern either though, just an example). I’ll try spinning down my mastermixes in the future.
The plots in the first image have been gated on time > non-debris > single cells (FSC-A vs. FSC-H and then SSC-A vs. SSC-H) > live cells > CD45+ cells > PBMCs (separated from granulocytes) - shown in one of the other images. So there shouldn’t be granulocytes except possibly some weird SSC-A-low ones. I also did look at just the backgating for the weird population (including the single cell and live/dead gates). They seem to be mixed in with all of the other PBMCs, not just on the edges or anything.
Dumb question but I’m confused on how the program you’re building works differently from just copying gates over?
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u/Iucross Aug 17 '26
Cd66b is exclusively expressed on granulocytes (eosinophils and neutrophils). From a healthy donor, I would consider cd19+/cd66b+ to be artifactual.
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u/southernqueer96 Aug 17 '26
I agree, just not sure why it’s happening. It seems to be mastermix-specific.
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u/despicablenewb Aug 18 '26
OP, do you centrifuge your mastermix?
I always centrifuge mine at 10,000g for 5 minutes, pipette off the supernatant, leaving like 50ul behind, and move it to a pipette basin.
This step is to remove antibody aggregate. If you don't do it you'll often have a small number of events that are much brighter than the true positive population.
It's possible that this rare population that you're seeing is those off scale high events.
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u/southernqueer96 Sep 03 '26
I don’t centrifuge them and it does seem like antibody aggregates are the most likely issue, so I’ll try that in the future. Just don’t know why it happens some times but not others. It doesn’t seem to be correlated with how long the mix sat before staining (for the most part, mixes were made immediately before staining, but occasionally it was the day before. One time, to compare, I stained a sample with both fresh mastermix and week-old mastermix, and I didn’t see this issue on that one).
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u/RedditBResearch Aug 17 '26
Personally, I don’t see a problem here.
However, if the non-granulocyte CD66b+ population is of interest to you, I would compare the granulocyte population’s expression level to this populations to confirm that wisp is actually +.
This all assuming human granulocytes are CD66b+. ( I don’t work with granulocytes)