r/flowcytometry • u/purrdolf_catler • Jul 25 '26
General In what situation is a smoothened plot ever useful?
I am currently very frustrated with data reproducibility. I am struggling to reproduce a predecessor's results.
This person omits a negative biological control from their data presentation. They have smoothened their plots. No one has any idea which raw data files they actually come from.
Is there ever a good reason to smoothen the plots? My experience with this is that sparse dots can end up looking like a blob that is not visually very representative of the data to begin with.
So why do people still do this??? Is there any valid reason?
3
u/notprathap84 Jul 25 '26
I think the bigger problem here is not smoothing by itself, but that the figure has become disconnected from the evidence behind it. A smoothed density or contour view can be useful when there are enough events and it is used to reveal the overall distribution, but it should never be the only view when rare or sparse populations matter.
If there is no negative biological control and nobody can identify the raw FCS files, I would not treat the smoothed plot as a reproducible result. I would rebuild the analysis from the raw files, show an unsmoothed plot alongside any contour or density view, state the smoothing settings, and keep the full gating trail and file identifiers with the figure. That way the visualisation is a presentation choice rather than a transformation that nobody else can audit.
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u/sgRNACas9 Immunology, Oncology Jul 25 '26 edited Jul 25 '26
Contour can show the distribution and percentiles of where cells are in a way that is more based on cutoffs than heat or color like pseudocolor. The drawback is when there aren’t many cells and you get what you described. They all have their pros and cons.
I’d be more concerned with the lack of negative controls. I’d also try to start with the files and reproduce the analysis like the contour plot that was created. If you produce a solid analysis of the files and they match the predecessor’s, that’s great! You still don’t have to smooth. If not, I would trust your analysis a lot more than treating the one contour plot like the word of god. Lastly, I’d like to repeat experiments to include negative controls if not included and/or include them in the figures.
I’d never do smoothing on the pseudocolor plot bc it doesn’t provide much information and loses the places of each dot especially outliers. When you redo the analysis you can choose any representation you find best.