There are many factors that can lead to the RBC contamination eg. low temperature of whole blood, whole blood that has been drawn for a long time, Hemolysis of blood, DEC that you use in the centrifuge. I am not so sure that after collecting the PBMCs, those cells are clumping or not?

For the cell counting with trypan blue, you can differentiate the morphology of PBMCs (mostly contain the granules) from the RBC (mostly plain and looks red). If you just freeze them to use later, the RBCs are mostly disappeared after thawing.

There is the product that could help - you can search Sepmate tube from Stemcell. It is easier than the conventional method but it's expensive.

Hello Thanh-Son Phan

1.

If you delay processing the blood samples, then over time, granulocytes and red blood cells in blood change density, and granulocytes degranulate. This means that processing older blood samples ( i.e., greater than 48 hours after blood collection) using density gradient centrifugation may result in granulocyte and RBC contamination.

In such a case, you will require longer centrifugation time and an additional RBC lysis step in order to reduce contamination. After isolating PBMCs, you may use RBC lysis buffer. For instance, lysis with ACK (ammonium-chloride-potassium) buffer can reduce the frequency of RBCs in the sample.

2.

RBCs do stain with trypan blue. As far as possible avoid RBC contamination while isolating PBMCs as it may lead to inaccurate counts. If you still get RBC contamination, you may prepare a 1:1 mixture of Trypan Blue and 2% acetic acid that can be used to dilute the PBMC cell suspension. The acetic acid will lyse red blood cells which may be present in the sample.

Best.

Hello Thanh-Son Phan ,

I've been isolating PBMCs using the gradient method for a while now and I also noticed that sometimes this happens. So far, I have no explanation for this phenomenon, meaning I did not notice any pattern. To me (and my team), it seems to occur randomly. However, this is quite rare. Therefore, if it happens often to you, then maybe there is some mistake during the isolation that you can identify and work on.

LATER EDIT regarding @Malcolm Nobre 's answer. I subscribe to what he said - keeping the blood for too long before isolation can do that. On the other hand, I would add that I have always processed fresh blood (2-4 hours from draw, sometimes up to 6 hours) and still this RBC contamination occurred (rarely, but it did). What is the time interval between blood draw and PBMC isolation in your experiments? How are you transporting and storing the blood during this time?

As Chutiphon Saelee said, there are many possible reasons and I subscribe to his answer. The centrifuge's deceleration setting is important. When I first learned this technique, the centrifuge had quite an abrupt breaking and because of that, the PBMC layer was spread all over the place, making it hard to recover without also recovering a lot of the gradient medium. However, I do not recall such RBC contamination upwards even when the braking was abrupt, so I would say this is not the case here. Especially since you said that it only happens with some samples. If the braking was the problem, then it should, theoretically, happen to all samples more or less.

I see you are using 50mL tubes. How much blood are you processing per subject? For troubleshooting, it may be helpful to divide a subject's blood in two 50mL tubes and see whether this occurs in both tubes of the same subject. If so, then it's more likely that the problem rests with the subject or with their sample (improper draw, transport, storage, waiting time etc). If the problem occurs only in 1 of the 2 samples of a subject, then it's most likely that this is a user-related problem. In this case, I can only say, from experience, that practice makes perfect. Keep isolating PBMCs and, at some point, some problems will just disappear even without you being able to pinpoint the exact cause.

As for differentiating RBCs, there was a similar question a while back. Take a look at these answers: https://www.researchgate.net/post/Does_the_RBCs_stained_with_Trypan_Blue_dye

Good luck!

Ion Bogdan Mănescu Malcolm Nobre Chutiphon Saelee

Thanks for the answers! Normally I process the fridge- stored(-4°C) blood within 5 hours after blood draw, therefore it cannot be the reason. Neither do I use a decceleration brake. Centrifugion is set on 600g for 20min

I use blood of patients that have acute-on-chronic-liver-failure, severe decompensated liver cirrhosis or after liver transplatation. Could that affect the quality of my ficoll gradient and result in clumping rbc?

Per 50ml Falcon Tube, I use 15ml Ficoll and 35ml mixed blood (patients blood+PBS, ratio depends on the success of blood draw and the patients fluid balance)

I try to avoid rbc lysis since it changes the protocol and interferes with the comparability of my data.