I am experiencing large variations in our Peripheral Blood Mono-nuclear Cell (PBMC) isolation from NHP whole blood. We are currently using Percoll diluted to 60 % to isolate, and the results have been very inconsistent. In some instances (second picture), after spinning (2000 rpm RT break off) I observe a clear separation of the 4 layers that is ideally seen, 1) densely pelleted RBCs, followed by 2) gradient, 3 )"buffy" white layer with PBMCs, and 4) wash media / plasma. In other instances, (first picture) the RBC pelleting is not complete and can be seen throughout the other layers. This requires additional lysis steps and also affects our isolation efficacy. I have not yet been able to determine any sort of correlation with NHP treatment and the varying results. Does anyone know what can be causing this?
Thank you for so much for the advice! I am a tech who receives the blood after it is drawn, so I am not sure how exactly it is drawn. However, it is stored typically at room temperature and on a slow shaker until I pick it up. Do you have any suggestions I can pass along to our surgeons as to how they should draw the blood to improve the quality of our samples?
No I do not travel far, it is just on the floor below me. Could it have anything to do with its time on the slow shaker? I typically process the blood right away after drawing, but some fellows let it sit overnight on the slow shaker before they begin to process. They are currently collecting them in syringes, however, so I will suggest we start to think about using vacutainers. Chris thank you again for all of your help!
Dear Josh. In general, any kind of stress can cause homeless, i.e. if blood is drawn to quick, if patient is too agitated (either in general or because surgeon had to pock them several times before having a good flow). I don't think there is anything wrong with your protocol, in our experience, it has more to do with the sampling per see. Was blood always drawn into vessels with the same anticoagulant? We sometimes see differences between heparin and EDTA.
Dirk,
Yes in our protocol now all blood is drawn into syringes with heparin as an anticoagulant, however, I am not sure how much is used per draw. We have been considering changing to EDTA pre-treated vacutainers, or keeping stock EDTA to use in place of heparin. In your experience, do you have better results with one anticoagulant over the other?
Also, we have been considering to routinely remove blood plasma prior to PBMC isolation; do you think this will have any effect on our isolation results?
In our hands, EDTA works better, but removing plasma has no effect. If you want to do it really fast, have a look:
http://www.stemcell.com/en/Products/Popular-Product-Lines/SepMate.aspx
These work really well
Dear your picture indicate that this due to the improper handling of the blood
1- check your EDTA tube provider and storage conditon or transportation condition
2- What is your centrifugation speed and time? if still this problem persist centrifuge it high speed and more time
2- If above condition are good, than what you are using for dilution of blood? normally Use DPBS
3- still have problem use ficoll instead of percoll. different companies provide ficoll specifically for the isolation of the PBMC, i am using ficoll and working good, (calculate the specific gravity of your percoll , may be problem with your percoll)
still have problem
collect blood, centrifuge it 800g for 10 minutes, remove the serum and collect the buffy coat ( WBC's), dilute with DPBS 1:2 and the layer on percoll or ficoll, you will get very clear PBMC,s. if some traces of RBC's then use RBC:s lysis buffer. wash it and use it. HAve fun
Dirk. Thank you all of the advice and for the product recommendation. I have looked into this product and when trying it out my isolation efficacy was poor due to the same problem I have described in my questions, where RBC pelleting was not complete and a significant "red" population was still observed after pouring off. I would ultimately like to switch our routine isolation protocol to use the sepmate tubes in conjunction with Ficoll once our sample qualities improve.
Ihtesham, Thank you aswell for the advice:
1- We currently use Heparin as an anticoagulant, and I will be trying to switch to EDTA as it has been suggested to be better.
2- Our normal protocol calls for 2000 RPM for 30 min at RT with the break off. It has been suggested that I can extend the time, but will increasing speed affect my isolation results?
2(b)- We either use a "Washing media" which includes is made up of 500 mL 0.9% Sodium Chloride, 2% FBS + 7 mL Pen/Step, or we use 1X DPBS with 2% FBS (Depending on downstream applications.
We also typically dilute 5-7 mL of blood in 20-25 mL of media. Other sources I have found suggest a 1:1, or 1:2 dilution of blood:media; could over dilution be affecting my isolation results?
3 - And yes, I have looked into Ficoll and other density gradients. I am worried that our 60% Percoll might not be dense enough for proper PBMC isolation.
Any input you have is greatly appreciated!
Josh, from all you write, I cant see a real reason why this happens. We normally spin blood 40min at 1200xg, so a lot harder than you do. We dilute bood 1:2 in PBS w/o Ca/Mg, and dont have any antibiotics in there. The only thing I can imagine is that there is something "wrong" with your washing buffer. But maybe it is just "one of these things" where individuals react different to the treatment?
Dirk, I have spoken with our surgeons and we agree that it may be due to poor sample quality resulting from our current blood drawing method, which calls for a syringe draw. We are going to be switching over to EDTA pre-treated tubes to see if this improves our sample quality, and subsequently our isolation results. I will also most likely be switching our washing media to supplemented PBS instead of saline.
As far as treatments are concerned, some animals do receive treatments that could affect RBC count (especially the ones that get irradiated) which I think could be affecting our results as well. However, in some monkeys that have received no treatment, I have observed the smearing as well, which again leads me to believe it is something to do with our sample quality. This week we are trying the EDTA pre-treated tubes and I will keep you posted as to how this affects my results!
Good. If you use syringes, don't pool to fast - you may have mechanical problems with too much vacuum. Can you use EDTA-coated vacationers? Might be an easy solution.
Just a side note - as for diluting the blood, in my experience more is always better. Our rule of thumb is at least 1:2 dilution of fresh blood (
Josh i suggest you, collect blood and centrifuge before addition of any thing, remove the buffy coat(WBCs). that will make almost 1ml of your whole blood , than dilute it and put it on percoll or ficoll, you will get very excellent result. have try. the whole blood thing, you always have contamination with RBCs, u tried alot but never get very good results.
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