Double CD8 population

[CRStevens]

Hi All,

I've noticed this a few times and can't figure out what makes this happen. I was hoping others in the community see it as well and can help me out here. I thawed 10million PBMCs and stained them with a really basic panel of T-cell markers (only like 8 markers) as I was just doing testing on some tetramer work we're working on. I noticed this strange double CD8 population and have seen it previously, but not sure what makes it happen as it seems random. There also seems to be some CD4 smearing as well. I show the CD8 vs time to show that these two populations are pretty consistent over time, so it doesn't seem like it is losing metal. I also noticed that overall my background seemed increased.

Do others ever see this double CD8 population? Is that a donor to donor thing or is it a bad staining issue? I've used this particular antibody many times and never had issues on other PBMC problems.

I also have recently have switched to WB injector and CAS... and have seen these 140's range metals coming off on long runs. I did have to run this sample for about 5-6hours, so was a longer run.

Thanks for any possible nuggets of information.

-Chad

[GregHopkins]

Hi Chad,

Are these cells from some conditions that may have a divergence between lots of CD8 effectors and CD8 naive memory cells? We have conditions in some of our assays that favor just naive/memory and others that favor a mixture - in the ones with a mixture, we often the CD8 double population. I think these cells fundamentally have different intensities/densities of CD8. Just a theory, but based on an observation from previous experience.

Greg

[JoDFCI]

Hi Chad

What do you have on your 146Nd

Jo

[CRStevens]

Great question Jo,

I do not have anything on 146. I have CD3 on 148Nd. I also did notice, though, that i have a signal in 163Dy next to CD8 and that channel is blank.

[JoDFCI]

check 162 on y vs 146 on y
and check against your monocyte marker
you might be surprised

[CRStevens]

Here is the 163Dy vs CD8

[CRStevens]

Thanks Jo,

I dont have any mono markers on this particular experiment, but will show what it looks like on CD3- population:
Dont see any issues on 146Dy

[nsigal]

Hi Chad,
Since there is nothing equially bright on other Dy channels and/or 146Nd then I think your CD8 is overtitrated and this is not second population but very high background due to overstaining. Maybe try to titrate it down and see if it helps? Also since you just switched to running samples in CAS and WB injector, please talk to your Fluidigm FAS about elevated background during prolonged sample acquisition in CAS. This information should be delivered to each and one of Helios CAS users!

[CRStevens]

I have spoken with them. I had previous issue with original CAS with 145 and 148 coming off on long runs, but have switched to CAS+ buffer and it seems to work well so far.

-Chad

[mleipold]

Hi Chad,

I think this might be biological variance. I'm not sure that I have seen CD8 "double" populations of quite your intensity, but I do see a downward "smear" or minor second population occasionally.

I have attached a PDF of 21 Healthy ctrls, gated on T cells (LiveIntactSingletsCD14negCD33negCD3pos; stained and run as one batch), plotted as CD4 vs CD8, and as a CD8 histogram. Some donors have essentially no CD8dim population, some have a bit of a smear down.

However, some donors like 424 (page 2, top row left), 447 (page 2, top row center), and 3369 (page 4, top left; final donor) has a clear CD8dim hump in the histogram.


Mike