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Changing % T cells in solid tumor model

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kurtOK

Participant

Posts: 1

Joined: Sun Aug 23, 2020 1:28 pm

Post Mon Dec 27, 2021 5:58 pm

Changing % T cells in solid tumor model

I'm a recent M.S. Applied Statistics grad on a steep CyTOF learning curve.

I was asked to append an existing Cytobank experiment with 9 additional samples. The 25 original samples are from 2020. The 9 new samples are from 2021. I used existing 2020 gates as a jumping off point for definition of tailored gates for the 9 new samples.

A prime focus of this experiment is the characterization of tumor infiltrating T cells in a solid tumor model. While the 2020 samples produced substantial numbers of T cells, the 2021 samples did not. Median in-gate percentages are shown below for the 10 cleanup gates and the gate for CD3+. In 2020 the CD3+ gate typically identified over one-third of cells in the Cleanup_DNA3 population as being T cells. In 2021 this identification rate was only 1 percent (median of results for 9 samples).

Or25 New9 Median % in Gate (Literal)
99 98 Median % in Cleanup_Ce140 on Ungated
97 88 Median % in Cleanup_Residual on Cleanup_Ce140
96 96 Median % in Cleanup_Center on Cleanup_Residual
97 96 Median % in Cleanup_Offset on Cleanup_Center
95 97 Median % in Cleanup_Width on Cleanup_Offset
99 98 Median % in Cleanup_EventLength on Cleanup_Width
44 100 SEE NOTE 1 Median % in CD45+ on Cleanup_EventLength
66 60 Median % in Cleanup_LiveDead on CD45+
81 72 Median % in Cleanup_DNA1-2 on Cleanup_LiveDead
95 95 Median % in Cleanup_DNA3 on Cleanup_DNA1
37 1 Median % in CD3+ on Cleanup_DNA3

My QUESTION is: What experimental or analysis issues are most likely to explain this large decrease in the percent of detected T cells? I presume there are dozens of possible factors and I’m hoping that others with more experience than me might help me prioritize my next troubleshooting steps.

NOTE 1. One difference between the processing of the Original 25 samples (2020) and the New 9 samples (2021) exists. In 2021 the EasySep Mouse TIL (CD 45) Positive Selection Kit was used to retain CD45+ cells (only) prior to treatment with CyTOF reagents. Permanent binding of EasySep antibody complexes to the CD45+ cells prevents the 89Y_CD45 reagent from binding to CD45+ cells, so 89Y_CD45 intensity levels are not useful (or necessary) for selecting CD45+ cells.

NOTE 2. An additional difference exists between the processing of the Original 25 samples (2020) and the New 9 samples (2021). In 2020 the samples were stained for viability with cisplatin and then frozen prior to being thawed and the rest of the staining completed for running on the Helios. In 2021 the samples were frozen and then thawed prior to cisplatin staining, and then immediately stained with the rest of the CyTOF reagents for running on the Helios.

The differences described above between the processing of the Original 25 samples (2020) and processing of New 9 samples (2021) were not expected to have any effects that would prevent our pooling the 34 samples. All other experimental conditions (e.g., treatments, mice) are believed to have been nearly identical in 2020 and 2021, conducted with the same reagents and personnel.
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antonio

Contributor

Posts: 20

Joined: Wed Dec 04, 2013 3:05 pm

Post Sun Jan 02, 2022 6:58 pm

Re: Changing % T cells in solid tumor model

Dear,
how can you be sure that "The differences described above between the processing of the Original 25 samples (2020) and processing of New 9 samples (2021) were not expected to have any effects that would prevent our pooling the 34 samples."? Did you test whether the EasySep Mouse TIL has an effect on other markers than CD45 in your experimental setting? Could you share some pictures or FCS?
Happy New Year!
Antonio
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mleipold

Guru

Posts: 5451

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed Jan 05, 2022 9:40 pm

Re: Changing % T cells in solid tumor model

Hi Kurt,

I would second Antonio's request for actual example bivariate plots for 2020 and 2021 samples....it's almost impossible to help you troubleshoot without pictures. Please start all the way at the top with Ungated, and show your plots as you gate down to CD3+...that way we can see how the cells changes as the gates are applied.

In particular, this will help us see how you adjusted the CD45 gate to deal with the EasySep antibody blockade of your probe CD45 (or if you took out that gate entirely).


Mike

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