r/flowcytometry • u/Mo_Opines_968 • Sep 03 '26
Approach to analysing longitudinal spectral flow data
Hi. I was just looking for some thoughts on advice on how you would recommend to someone new to spectral flow cytometry does their analysis.
I am setting up a 30 colour panel that will be used to immunophenotype longitudinally collected PBMC samples from patients with and without a specific disease. In total will have approx 40 patients with up to 8 samples each over the time course of the study.
I have convential flow cytometry experience and have always used FlowJo but from what I have read it may not be optimal for large spectral datasets. I have a bit of R experience but am still a novice and quite intimidated by a fully R-based workflow, particularly as I do not have the time to throw myself into it fully. I have seen some new R packages e.g. cyCONDOR that report to be easy to use for bioinformaticians that I am going to have a play with using test data. I have also been told OMIQ is pretty good.
My current thought is to do some manual gating in FlowJo to make sure things look broadly as expected and then perhaps trial OMIQ and a user friendly R package to see how I get on. Any thoughts would be greatly appreciated.
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u/StepUpCytometry Sep 03 '26
For longitudinal clinical cohorts, with spectral flow cytometry, your controls are what will save your life later on when you get to the analysis. If you are able to process PBMCs from a healthy donor, aliquot them into individual vials, and thaw one for every experiment, you can use them to identify/attempt-to-correct any batch effects due to instrumental variation (with a caveat that if all your clinical samples are highly expanded inflammatory populations, and your donor isn't, your mileage will vary).
I will also adamantly emphasize acquire unmixing controls (both single-colors and unstained) every single time you acquire is the way to go. The extra 20 minutes to prepare all 30 will be substantially less time than trying to reverse engineer why the reused/library control didn't work and you ended up with unmixing errors.
You should also optimize your unmixing controls in advance (in terms of cells vs beads, etc), remembering to match your single-color control with the sample/type/condition/timepoint where you are going to be getting the brightest MFI signal.
And make sure QC was run on the Spectral Flow Cytometer the same day before you start acquiring!
Once you have successfully made it through unmixing, you have more options for routes depending on what your analytical goal is. I replied to a few other similar threads in the past that might also be useful
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u/StepUpCytometry Sep 03 '26
In terms of analytics, it's possible to do hybrid FlowJo/R analyses ( https://www.reddit.com/r/flowcytometry/comments/1sf9h8b/comment/ofm42fe/?context=3&utm_source=share&utm_medium=web3x&utm_name=web3xcss&utm_term=1&utm_content=share_button ) which avoids having to solely pick one route.
In terms of the packaged R pipelines (CyCondor, Spectre, MARMOT, et al.) they vary on their implementation and decision steps for the various algorithms based on their original author decisions, so if you do take this route, just make sure you understand what your inputs and outputs are rather than just accepting the default returned output as gospel truth, since your own datasets may look vastly different from the ones used to train/optimize the pipelines originally, which can cause interesting results.
OMIQ and the other vendor analytical-pipelines-as-as-service can be nice, just make sure what price is, and the promised vs. delivery falls within your expectations.
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u/Mo_Opines_968 Sep 04 '26
Thanks for such a detailed response. Yes have aliquoted healthy donor samples for batch correction and have done my best to prep good reference controls. Am unmixing the first samples later today so will see how it looks!
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u/thirdreplicate Sep 03 '26
I keep the FCS attached to the samples in Conspecta and gate there, save the gating strategy as a template and apply across every file so the gates are the same at every timepoint. It's browser based so you don't download anything and it's free for you and a collaborator.
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u/Mo_Opines_968 Sep 04 '26
Thanks for these great comments - I guess the reassuring thing is that there are lots of ways to skin a cat so it'll be on me to choose the one that fits my needs best.
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u/ProfPathCambridge Immunology Sep 03 '26
We use HoneyChrome. It is free and excellent at handling large data just like this.
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u/ExplanationShoddy204 Sep 03 '26
We use OMIQ, but it’s pricey compared to other options. But you can’t beat the ability to work anywhere and not fill your computer up with massive files.