Hello Lorenzo Tala,

There is no way to extract serum and PBMCs simultaneously from a single blood sample though there is one study which I came across that claims to have developed a protocol to isolate lymphocytes from clotted blood. You may want to refer to the article attached below for more information.

https://doi.org/10.1016/0022-1759(87)90408-x

I would suggest the use of plasma instead of serum as sample type to test cytokines and circulating antibodies because cytokine levels in plasma are more stable compared with serum. Plasma is a better matrix than serum.

Moreover, non-specific background is significantly increased in serum and as a result of this, disease-related decreases in low-abundance cytokines can sometimes be detected in plasma but not in serum.

Also, serum is the soluble part of clotted blood and is obtained following blood coagulation. Blood cells may be activated during the clot formation and as a result cytokines may be released from platelets into the serum.

Plasma, on the other hand, is first treated with anticoagulant before the blood cells are removed by centrifugation, so that clotting factors remain deactivated in the liquid.

If you decide to use plasma, then you may use the method described by Lyudmila Akhmaltdinova, and you could minimize sample collection from patients.

You may have the opinion of experts in this field and decide for yourself the choice of sample type.

Best.

Hi Lorenzo Talà

Here are some of the protocols I found could be of help.

1. Density gradient centrifugation:

• This is the most widely used method, separating blood components based on their density. Blood is layered on a density gradient medium (e.g., Ficoll-Paque), centrifuged, and distinct layers containing plasma, PBMCs, and red blood cells are collected.
• Advantages: Well-established technique, relatively high yield of both serum and PBMCs.
• Disadvantages: Requires specific equipment and reagents, longer processing time compared to some other methods.

2. Single-spin gradient centrifugation:

• This is a faster variation of the above method, using pre-filled gradient tubes and shorter centrifugation times.
• Advantages: Faster processing, readily available kits.
• Disadvantages: Yield might be slightly lower compared to traditional density gradient methods.

3. Immunomagnetic separation:

• This method employs specific antibodies targeting PBMC surface markers to magnetically isolate them from whole blood.
• Advantages: High purity of PBMCs, enrichment for specific subpopulations possible.
• Disadvantages: Can be more expensive compared to other methods, potentially lower overall yield.

4. VACUETTE® CPT™ tubes:

• These pre-filled tubes contain a gel barrier that separates plasma and PBMCs during centrifugation.
• Advantages: Convenient and easy to use, good separation of plasma and PBMCs.
• Disadvantages: Limited volume capacity, potentially lower PBMC yield compared to other methods.

Thanks

Thank you very much for your reply, this is super useful!!

I have worked with serum in the past and did have hard time getting consistent cytokine levels with ELISA. So I'll definitely try with the plasma.

Thanks again for the papers and protocols. I'll try this out.