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Red blood cells in ELISpot

Guest LRS

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Do red blood cells interfere with ELISpot assays? Is there any evidence that have systematically tested this? Do you have any suggestions on how to test this? Will the contamination of red blood cells affect the ELISpot results?

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Generally a limited amount of erythrocytes is likely to have little or no effect on ELISpot results. However, as the assay is dependent on the cytokine-secreting cells being in relatively close contact with the antibody coated membrane and erythrocytes may compete for space, there is a limit to how much erythrocytes you can have before they start to interfere. So, even if it - in most cases - would work just fine, the risk is that you will have varying amounts of “contaminating” erythrocytes in different cell preparations and therefore it may be safest to still try to remove them. Apart from lysing the erythrocytes, which most seem to do even though it’s been reported to give varying results, people use Ficoll centrifugation which is probably a better way of doing it.    


Kind regards,



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  • 4 weeks later...

Hi Lauren and Julia,


I would just like to give this thread some nuances.


We have tested running whole blood using our standard protocol (as if we would run PBMCs) and it does not work well. Besides the problems Julia mentioned (the red blood blood cells are just so numerous, they block the way for the analyte to reach the capture antibodies), there will also be a lot of background coloring due to the blood which makes it hard to analyse possible spots.

I’m not aware of any systematic testing that’s been done to show at what concentration of red blood cells they start to interfere. But theoretically, one could imagine using separated pure PBMCs and then adding increasing amounts of Ficoll pellet (containing red blood cells and granulocytes) and study at what point problems arise. This would give some information on how many contaminating erythrocytes one can accept. Once there, one could take the sample where problems began and measure the haemoglobin value. That value could then be related to a “real” sample, to be able to say “ok, the haemoglobin value in this sample is under X, then it’s ok to run the assay”.  But as said, we have not tried this in-house. Instead, to be on the safe side, we recommend doing a Ficoll separation, because then you know there will be minimal amounts of red blood cells there. An alternative or complement to Ficoll is a red blood cell lysis protocol. With some samples there is a risk of getting remaining contaminating red blood even after a Ficoll. I used to work with tonsils back in the day, and the PBMC band was always super red after Ficoll separation. I then complemented with a few minutes of red blood cell lysis solution which gave me a totally pure PBMC preparation. But if you’re working with fresh blood, there should be no need for other methods than Ficoll.

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  • 8 months later...
Guest Lily

Hi,  has anyone tried to lyse red blood cells in whole blood then wash and use it straight away in ELISPOT assay? anyone knows published papers describing the effect or red blood cells on ELISPOT?





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Hi Lily,

I hope another Forum-user than me can shed light on your quest for hard evidence papers showing the effect of RBC on ELISpot. As I wrote above, there are theoretical ways of investigating this, but I have yet to see them published in practice.

But regardless, for sure, an RBC lysis of whole blood before an ELISpot assay could work out ok, and we have performed it occasionally in-house. But after talking to some of our veteran ELISpot experts here at Mabtech, our official answer is that we would advice against RBC lysis and thus still recommend the Ficoll separation method. Because: with Ficoll you also get rid of most granulocytes. 

Granulocytes may, just like Julia writes above for contaminating RBC, compete for space on the PVDF membrane, perhaps reducing the contact between e.g. APCs and T cells and thus lowering the sensitivity of the assay.

In addition, contaminating (and activated) granulocytes may directly interfere with T cells, reducing their CD3z-chain expression, especially in blood stored for a long time before PBMC separation. I made a small "review" on the literature surrounding granulocyte contamination and how to avoid it, and the take-home message from that literature overview is that it if you're not preparing PBMCs from the blood immediately after collection, it is recommended to dilute the blood 1:1 in PBS or medium before storage. By an unclear mechanism, the dilution procedure reduces the negative effect that activated granuolcytes may have on T cell responses (at least true for IFN-gamma). I am attaching the literature overview here in its entirety, but please note that the content of this ppt-file is my personal take on the issue, and not an official Mabtech document.

Granulocyte contamination in ELISpot.pptx

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