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Batch Controls

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bakerk

Participant

Posts: 2

Joined: Thu May 19, 2022 5:39 pm

Post Tue May 31, 2022 11:15 pm

Batch Controls

Hi everyone,

I am looking to see what others do to control for batch variation. I have a couple big projects coming up that will be split into several batches. My samples are frozen PBMCs and I will be running a custom panel on them (MDIPA kit plus some custom such as FoxP3, Ki67, Granzyme B) that uses intracellular staining. I currently do not implement any barcoding.

Is my best option to buy a large quantity of fresh PBMCs and aliquot it myself into ~3 million cell vials to run with each batch? Does anyone have a preferred vendor? I have only looked into AllCells and they have told me 100M is their smallest size for fresh.

Also, since I am using intracellular staining, would I need to create a combination of stimulated and unstimulated cells? This seems like it would be difficult to do. Considering cost and time, I think I will realistically only be able to run one control sample per batch.

I haven't tested it out yet, but am planning on using CytoNorm (Spectre) for batch alignment.

Any advice would be helpful, thanks!!
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MCOlivier

Contributor

Posts: 44

Joined: Mon Oct 05, 2015 9:48 am

Location: European Genomic Institute for Diabetes, Universitity of Lille, Institut Pasteur de Lille, France

Post Wed Jun 01, 2022 6:55 am

Re: Batch Controls

Dear Bakerk,

Many points raised here :)

First of all, I strongly advise you to barecode your samples, in order to get real batches, like one pool/batch of 19 samples + THE control per day.

For THE controls included in each batch, use PBMC from maybe one of your collaborators or a control healthy individual punctured with a high volume in order to get enought PBMCs/aliquots for you whole exepriment (of course 1 aliquote per batch).

In my hands, Granzyme B does not require PMA/Iono and Monencine/brefeldine exocytosys blockade to get good signals in CD8/NK cells. So I would advise to avoid a stim/blockade step, in which you will loose a lot of cells.

For Batch adjusment, we are going to release a user friendly update of the Batch Adjust package published by Schuyler et al. in a few weeks, and basically I prefer Batch Adjust to Batch Norm package, because Batch Norm package requires pre-gating of cells, introducing what I would consider as a bias in many channels correction because of the population-specificity, like why correcting differently Granzyme B in CD8 and NK cells, making their "standard" levels potentially different in these populations and consequently avoiding comparison of GrB levels between those populations ?

Last, avoid also quantile methods, because of their non linear correction, potentially introducing bias and artefacts by shifting cells from "positive pike" to negative pike", depending on proportions of cells in those + or - pikes (sees Schuyler et al publication).

Best regards ;-)
Olivier

Schuyler et al :
https://doi.org/10.3389/fimmu.2019.02367
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sgranjeaud

Master

Posts: 121

Joined: Wed Dec 21, 2016 9:22 pm

Location: Marseille, France

Post Wed Jun 01, 2022 8:46 am

Re: Batch Controls

Hi,

From discussions with experimenters, I would recommend a stimulated control if you hesitate between unstim and stim. The goal of batch normalisation is to align the positive peak of a channel across batches.

Best,
Samuel
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MCOlivier

Contributor

Posts: 44

Joined: Mon Oct 05, 2015 9:48 am

Location: European Genomic Institute for Diabetes, Universitity of Lille, Institut Pasteur de Lille, France

Post Wed Jun 01, 2022 9:54 am

Re: Batch Controls

Hi dear Samuel :),

Totally agreed for cytokines or any target that has to be stimulated to be "seen".
Concerning the 3 targets named by Bakerk : FoxP3 and Ki67 do not require stimulation. To my knowledge, GranzymeB does not require stimulation to be identified, as not processed intracellularly as cytokines, ie accumulated in granules for stimulation induced released.
As an illustration, look at the pdf attached in CD8+ T cells from PBMCs showing in parallel GranzymeB and Perforin (the last one being undetectable without stimulation).

Best regards ;-)
Olivier
Attachments
Oliviers new illustration.pdf
(57.15 KiB) Downloaded 108 times
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CRStevens

Master

Posts: 56

Joined: Thu Jul 17, 2014 5:07 pm

Post Wed Jun 01, 2022 2:47 pm

Re: Batch Controls

Bakerk,

If you still have the ability to do barcoding. I also highly recommend it. For large studies we tend to stain the samples fresh, and barcode at the IR intercalator incubation at the end so we can at least run all our samples in a single tube. We have also multiplexed this by using CD45 live barcoding as well. So the first 20 samples we stain with 89Y CD45, then the next 20 samples will be stained with CD45 194Pt, ect. This way we can manually pull batches out after the fact, but can run 40+ samples in a single run. We also spike in a control cell to normalize for staining batch effects.

As a note. If you are getting samples over a period of time. We live stain, fix and freeze at -80 for up to a couple months without any issues, so we can thaw all samples on one day, barcode and run that same day. This of course, comes with the caveat that your runs will be much longer and you'll have to monitor your signal doesn't get too low. Sometimes mid-run tuning is necessary.

-Chad
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mleipold

Guru

Posts: 5428

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed Jun 01, 2022 2:51 pm

Re: Batch Controls

Hi all,

I would point out that there are a few reasons to have controls in your experiments.

1. Make sure all your *probes/antibodies* are working properly (including whether you accidentally left out one of the 30-40 antibodies when making the cocktail.....). In this case, a single control (Unstim, or a single Stim) might be sufficient per batch, depending on your experiment. And, if your probes aren't sensitive to fix, you may even be able to pre-make Unstim and Stim cell aliquots so that you'd have enough for an entire study. I think we've had a study where the researchers got Biolegend to make Fixed Unstim and Fixed Stim Vericells that were enough for an entire study.....you might reach out to your rep.

2. Make sure that all your *reagents* are working properly. I've seen cases where a stock of PMA or LPS was working fine one week, and then something happened that caused it to stop working properly. In this case, if you were relying on pre-made Stim samples, you might not catch that your Stim reagents stopped working properly.


Therefore, in an ideal setting, you would have a control donor (PBMCs, or whatever your study samples are) that you treat exactly the same as all your study samples for *each staining set*: thawing, washing, counting, any rests or stims, etc. If you're running individual samples and not freezing them at some point, you may wind up running proportionately more controls than you would if you were doing larger barcoded sets (particularly if you were doing FBS/DMSO freezing at the end so you could stain multiple Plates in one experimental Batch).

However, there may still be ways to cut down on the total number of controls: for example, we've run some PhosphoPBMC sets where we thawed enough samples in one to fill two plates, but only did one control set. In this case, we did everything in parallel for the two plates up through and including the MeOH at -80C, and finished processing each plate separately. Formally, this only controlled for the stims (since both plates were stimmed together), not for the antibodies (at least the intracellular were stained in separate plates), but was considered to be a happy medium.


Mike
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MayaBr

Participant

Posts: 3

Joined: Wed Nov 11, 2020 8:32 am

Post Thu Jun 02, 2022 6:18 am

Re: Batch Controls

Hi Chad,

How are you freezing the cells after staining for long term storage?

Best,
Maya
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bakerk

Participant

Posts: 2

Joined: Thu May 19, 2022 5:39 pm

Post Thu Jun 09, 2022 11:34 pm

Re: Batch Controls

Thanks for everyone’s responses, lots to consider! Better examples of markers in my panel that need stimulation are Tim-3, Lag3, GATA3, and CTLA4. For these would you all suggest stimulation? I have done anti-CD3/anti-CD28 stimulation for them in the past for titrating. From my understanding, it seems all three of the main batch alignment methods (CytoNorm, CytofBatchAdjust, and CytofRUV) would require a reference population for each marker, meaning healthy PBMCs won’t be able to capture these.

Our samples get sent to us as frozen, unstained PBMCs, so I am unable to do any fresh staining or freezing by batch. This is also partly why I haven’t done barcoding previously, since the live cell barcoding wouldn’t account for variation introduced in staining.

Olivier- thanks for the paper and your explanation, I will keep an eye out for the update. Do you have any thoughts on Batch Adjust vs CytofRUV?

Totally agree with Mike that two controls would be ideal, one stained day of and one stained previously in a large batch. Maybe once I’ve found ways to increase my sample throughput I’ll have more time available for adding another control. Your “happy medium” suggestion sounds useful as well.

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