Time dependent changes in the bioenergetics of PBMCs: processing time, collection tubes and cryopreservation effects 2023 Werner et al

Thanks @Andy, this is a really important issue for all of the studies that have been done using the Seahorse analyser. We've been calling for information on this.

The paper describes the Seahorse analysis in a readable way and gives clear definitions of the parameters reported

It is good to hear that the results from the Seahorse analyser were found to be well replicated when the samples tested were identical (although I didn't see any results (charts) to support this).

The researchers did a lot of experiments, looking at storage time, tube type, ice type, effect of plasma manipulation, effect of cryopreservation:

I'd appreciate the views of members who have worked with the seahorse analyser and/or blood samples on the robustness of this paper. It looks good to me, probably mostly based on the clarity of the writing, but I haven't looked in detail and I don't have any expertise in this. It looks as though it would have been good to have more testing done during the period between 6 hours and 24 hours, and I would have liked to see analysis of the exact same samples over time.

Figure 2c suggests to me that for cell viability assessments, ideally there's a 4 hour window for assessment.



For the respiratory parameters, it's not so much the mean changes, although they do, it's that variability is lost. See, for example, Fig 3d for reserve capacity:



On long term storage methods for viability, even the best methods are affecting viability - see Fig 5A. There are other analyses there in Figure 5 for the respiratory parameters after long term storage.

 

So, keeping storage times to an absolute minimum, reducing variation in this and detailed reporting. Also, EDTA tubes were recommended.

I think this study raises some important questions for biobanks. I think they need to be very selective about what sort of analyses they supply stored blood samples for. Otherwise, they are just contributing to research noise, rather than useful results. If I was doing a seahorse analyser study on unmodified blood cells, I think I would want to be doing the analysis immediately if possible and certainly within 4 hours. That implies that biobanks need to have a registry of donors available to give blood directly for these sorts of studies.

I'm not sure what this means for studies where blood cells are converted into lymphoblasts. @DMissa, I'd appreciate your thoughts about this.

 

Also, with the recent WASF3 study in mind, we need a similar sort of study on the effect of standard processing and storage on other types of cells.