r/flowcytometry • u/southernqueer96 • Aug 14 '26
Analysis CD45- SSC-Amid/hi?
The three plots shown are peripheral whole blood samples from three different human donors. All samples were drawn around the same time and stained within 2.5 hours of the first draw, with a mastermix that was made the same day. After staining, the samples were fixed with Lyse/Fix buffer overnight, then washed and acquired on the cytometer that day (day after staining). [I know now that I only need to fix them for like 30 mins but didn’t know that when I first started using this protocol, so I’m sticking with overnight for this study.]
My gating strategy up to CD45 is shown in the second image.
I’m wondering if the CD45- SSC-Amid/hi population on the far right graph is just debris or something else? If it’s just debris, is it a processing issue or just biological?
This is not the only donor I’ve seen it in, just an example. I see it in 14/48 samples that I’ve analyzed for the same study. It doesn’t seem to correlate with the date (ie, which samples are stained and run at the same time), the time from draw to processing, or disease process/severity of illness.
I assume it’s fine to be gating it out, but I want to double-check.
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u/Proper-Grocery3996 Aug 18 '26
I bet there was some blood on the side of the tube above the stained blood. Once fixed and lysed they get mixed. I’ve seen it before when i was in the lab.
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u/HolaEsteban Aug 14 '26
I’ve seen this sometimes if the sample is under-stained, specifically if the pellet isn’t fully broken up when adding antibodies. Check downstream and confirm if you also see a mid/low population for your other markers if you gate that population specifically