Hi guys,
So I'm receiving blood samples each day so that I can harvest PBMCs for flow cytometric analysis (and neutrophils for possible DNA extraction). The diagnosticians that take the blood for us do so with green top tubes (containing lithium heparin).
Just wondering how long I can leave these blood samples at room temperature before PBMC quality and quantity is dramatically effected? I could process the blood samples each day, but I'm only getting samples from one individual per day and it would be much more efficient for me to process them all on the same day (i.e. once a week or so).
Any help or advice would be appreciated.
Thanks!
Dear David,
I would strongly recommend to analyse your samples on the same day within 6h after sampling. We have conducted several experiments in order to adress this question: granulocyte counts go down rapidly within the first 24h. T and B lymphocyte counts are stable for up to 3 days, whereas monocytes and NK cell counts decrease slowly after 24h. As for stimulation/proliferation experiments, you have to use the cells asap, because in whole blood dropping glucose levels (~10%/h) together with acidification harm the cells permanently.
Kind regards,
Werner
Neutrophils are pretty short lived, so if you are interested in those, you will want to process them the same day. How will you be isolating them?
About the PBMCs, I have tested isolation after 1, 2 and 4 days. Or at least, I meant to. After two days, the yield was so low that I didn't bother with the 4 day sample. The effect of storage at RT on the 'quality' (I suppose some activation markers) really depends on your situation.
I had the same problem and just ended up isolating cells from 1-2 patients per day. If you pre-pipette everything for e.g. 20 isolations, then it's just a matter of putting the blood into your ready pipeline. It saves a bit of time and makes your days easier.
Dear David,
I would strongly recommend to analyse your samples on the same day within 6h after sampling. We have conducted several experiments in order to adress this question: granulocyte counts go down rapidly within the first 24h. T and B lymphocyte counts are stable for up to 3 days, whereas monocytes and NK cell counts decrease slowly after 24h. As for stimulation/proliferation experiments, you have to use the cells asap, because in whole blood dropping glucose levels (~10%/h) together with acidification harm the cells permanently.
Kind regards,
Werner
Dear David,
As far as I know its better to isolate and do your experiment on the same day. However, I left the blood for 24h and then started to do the isolation.. It was fine because I focused on T lymphocytes and also monocytes.
Bests
Nazanin
My advice is that you process your samples on the same day. You will start having a decrease in granulocytes within a few hours and after 18-24h this could become significant. I am not ceertain that you would get any better cell survival outcomes with other anticoagulants like EDTA, but depending on what you intend to do with the cells heparin might not be the best choice. Bottom line is that the sooner you can process your samples, all the better. You can cut down on the daily hassle by setting up all your reagents in batch form so that you will just need to add your cells.
Thanks everyone for your answers - I ended up learning the hard way not to wait as long as 24 hours at RT. I attempted to separate the blood on a density gradient, and it would have taken several hours for the cells to separate properly, whereas fresh blood (on our density gradient) produces a neutrophil band and a PBMC band after a 30 min spin.
Regarding the EDTA tubes, perhaps these would be better, but previous experience in our lab has lead to successful isolation of DNA after neutrophil harvesting from lithium heparinised blood. Perhaps to do with our density gradient (made in house) or that we store our neutrophils as pellets at -80C? Not sure.
Thanks again guys.
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