Concerns about populations during manual gating

[DanielUMA]

Dear CyTOForum community,

I am performing data analysis of clinical samples from whole blood, stained with the Maxpar Direct Immune Profiling Assay (MDIPA) kit. After performing the scaling check, manual clean-up and quality control (PeacoQC), I have a some of questions related to the manual gating strategy. When representing CD45+ vs CD66b, in several samples there are two populations that I don't know exactly where to include:

1) There is a population that is slightly positive for CD45, it is not CD66b+, but it does not express any other marker clearly either. The only marker it expresses is CCR4+. I thought it might be an artefact, but it is quite a large population. https://imgur.com/a/cd45-low-population-0NFiTlX
2) The second question is about another population, also representing CD45 vs CD66b. In some samples there is a population very positive for CD45, but also expressing CD66b and CD16. Should I include them in the granulocyte gate or the PBMCs? https://imgur.com/a/uTq3jCS
3) Finally, I find that in some samples, the CD45+ population is positive for CD16. I find this strange because, although some CD45 cells express CD16 (such as monocytes and NK cells), I find that also for these samples T or B lymphocytes seem to express this marker. Could this be an artefact, and how could I correct it? https://imgur.com/a/Z7XXSfe

Thank you in advance!
Daniel.

[mleipold]

Hi Daniel,

A few initial questions:

1. When you say these are clinical samples, what do you mean? Are these just samples from healthy patients, or are they Cases with some condition or treatment?
- in particular, you say that this occurs "in several samples": is there any difference (Case vs Control, treatment, type of draw, etc) between samples where you see these issues and samples where you do *not* see them?

2. Have you tried putting these samples through the Pathsetter program? If so, how did these weird cells wind up? For example, if there's some kind of major artifact in the data, then you're likely to wind up with a lot of cells in the "dump" population (I forget the exact name, something like "Undetermined" or "Unspecified".....something that doesn't fit into the regular categories as determined by Pathsetter).

3. To confirm: when you say that you're staining WB, you mean that fresh (live) complete WB (including serum and RBCs) is being added to the MDIPA tube? Rather than fixed WB, or live but RBC lysed and washed?


Mike

[DanielUMA]

Hi Mike,

Thank you for your interest and kind reply.

1) I work with samples from both healthy individuals and patients. This issue with this population CD45+ happens for several samples including controls and patients. It isn’t a batch effect, as it also happens for samples adquired in different days.

2) that was a good idea. I passed some of this samples through Pathsetter, and the software didn’t consider this population as an artifact. It stays after the clean-up.

3) I am staining fresh whole blood, so the protocol includes adding the fresh blood (live and with RBC) to the MDIPA tube, and performing the lysis of the RBC right after.

Thank you,
Best regards
Daniel

[mleipold]

Hi Daniel,

Thanks for the answers.

I know that early on, there were some issues with MDIPA and some weird donor-specific staining (but only in WB, not PBMC, as it was a plasma-driven issue): https://www.biorxiv.org/content/10.1101 ... 0.405027v1

My recollection is that Fluidigm/SBio was eventually able to figure out what was going on, and supposedly addressed the issue. Among other things, I think they had to find a donor to draw for their testing, since not all donors had the issue (but if one did, that donor would reproducibly stain weirdly).


These aren't the antibodies you were mentioning, so I don't think it will be the same issue, per se. Is there any chance you could stain an affected donor again, and see whether it's reproducible, rather than random?

And, are all the affected donors stained with the same MDIPA lot, or different lots?


Mike

[DanielUMA]

Hi Mike,

Thanks for de information. I checked the issue about the early problems of MDIPA. It was reffered to unespecific CD19 staining. However, in my samples, CD19 seems to work well. Besides, the issue I observe with this big population of CD45+(low)/CCR4+ appears in both old and new samples, on which different lots of MDIPA were used.

As I have already finished the project, I don't have any more MDIPA tubes to repeat the staining with a donnor on which I observed this population, so I am not able to check if it is donnor-specific reproducible.

This population represents about 1/3 of all CD45+ in some samples. However, in other samples this population does not exist. My main concern is that, by including it in the gates and clustering analyses, I am inducing differences that are not real.

Thanks a lot for your help,
Daniel.

[ChantelMcS]

Daniel, Hi! Say, the question of CD16+ CD66+ population interested me. I think it is probably real. Here is an article (thank you, Google) that supports the idea that these are neutrophils.
"Imaging Inflammation in Asthma: Real Time, Differential Tracking of Human Neutrophil and Eosinophil Migration in Allergen Challenged, Atopic Asthmatics in Vivo"

Regarding the CD45lowCD194+ I think the they are basophils because according to Biolegend CD194 human is present on "B cells, Basophils, Embryonic Stem Cells, Monocytes, NK cells, T cells, Tregs". Also basophils are low expressing CD45.

Hope this helps in your search for answers ! Chantel McSkimming