Impact of Long-Term Cryopreservation on Blood Immune Cell Markers in ME/CFS: Implications for Biomarker Discovery Gomez-Mora et al 2020

[John Mac]

Full title:
Impact of Long-Term Cryopreservation on Blood Immune Cell Markers in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: Implications for Biomarker Discovery

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a complex neuroimmune disorder characterized by numerous symptoms of unknown etiology.

The ME/CFS immune markers reported so far have failed to generate a clinical consensus, perhaps partly due to the limitations of biospecimen biobanking.

To address this issue, we performed a comparative analysis of the impact of long-term biobanking on previously identified immune markers and also explored additional potential immune markers linked to infection in ME/CFS.

A correlation analysis of marker cryostability across immune cell subsets based on flow cytometry immunophenotyping of fresh blood and frozen PBMC samples collected from individuals with ME/CFS (n = 18) and matched healthy controls (n = 18) was performed.

The functionality of biobanked samples was assessed on the basis of cytokine production assay after stimulation of frozen PBMCs.

T cell markers defining Treg subsets and the expression of surface glycoprotein CD56 in T cells and the frequency of the effector CD8 T cells, together with CD57 expression in NK cells, appeared unaltered by biobanking.

By contrast, NK cell markers CD25 and CD69 were notably increased, and NKp46 expression markedly reduced, by long-term cryopreservation and thawing.

Further exploration of Treg and NK cell subsets failed to identify significant differences between ME/CFS patients and healthy controls in terms of biobanked PBMCs.

Our findings show that some of the previously identified immune markers in T and NK cell subsets become unstable after cell biobanking, thus limiting their use in further immunophenotyping studies for ME/CFS.

These data are potentially relevant for future multisite intervention studies and cooperative projects for biomarker discovery using ME/CFS biobanked samples.

Further studies are needed to develop novel tools for the assessment of biomarker stability in cryopreserved immune cells from people with ME/CFS.

https://www.frontiersin.org/articles/10.3389/fimmu.2020.582330/full

 

[ME/CFS Skeptic]

Merged post

If I understand correctly, this Spanish study looked at freezing cells might cause differences in immune markers compared to fresh blood.

Some of the most tested immune markers in ME/CFS remained stable when frozen, namely: CD57 in NK cells, CD8+ effector T cells, CD56 expression in T cells, and Treg frequency.

In contrast, other markers showed inconsistent expression in biobanked PBMCs when compared to their fresh counterparts: namely NKp46, CD69 and CD25 expression on NK cells.

A previous study by this research team reported upregulated NKp46 in fresh blood. The authors now argue that frozen samples from a biobank are not appropriate to replicate their results. They write:

The lack of correlation between NKp46 values in fresh and frozen ME/CFS samples (Figure 3C) further argues against future attempts to validate markers in samples other than fresh blood.

 

[cassava7]

This article seems to have worrying implications because, earlier this year, some ME/CFS research teams had to freeze fresh blood samples before their labs shut down due to the COVID19 pandemic (possibly still ongoing in some places). The Cornell ENID Center, who are studying immune dysfunction in ME/CFS with 2-day CPETs, have done so:


For instance, would freezing affect cytokines? I recall that the Stanford team who looked at cytokine profiles in ME/CFS (2016) went to great lengths to process their samples as quickly and as carefully as possible.

 

[Andy]

I know Karl Morten has concerns about the effect that storage might have on samples and results of analysis, not just with freezing but between storage mediums as well.