Thesis Characterising the Electrophysiological Properties of Cells in Health and Disease [on ME/CFS], 2024, Clarke

[Dolphin]

https://openresearch.surrey.ac.uk/e...siological-Properties-of-Cells/99893266002346


Doctoral Thesis
Characterising the Electrophysiological Properties of Cells in Health and Disease

Krista Samantha Pauline Clarke
University of Surrey
Doctor of Philosophy (PhD), University of Surrey
28/06/2024
DOI:
https://doi.org/10.15126/thesis.901143




Abstract
Dielectrophoresis Myalgic Encephalomyelitis/ Chronic Fatigue Syndrome SARS-CoV-2 PBMC COVID-19 Chondrocyte Diagnosis

Biological cells possess intrinsic electrophysiological properties which are fundamental to cellular function. Changes in cell electrophysiology can act as a biomarker, for example to indicate transition from healthy to diseased cell states, changes in cell function, or cell differentiation. This thesis presents three studies which used dielectrophoresis (DEPtech 3DEP) and ζ-potential analysis (two fast, label-free, high-throughput, non-invasive, and low-cost tools) to examine the electrophysiological properties of two cell types, peripheral blood mononuclear cells (PBMCs) and chondrocytes, for novel medical applications.

The first study investigated the electrophysiological properties of PBMCs in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS); a debilitating disease of unknown pathophysiology with no reliable, validated, and quantitative diagnostic test. The dielectric and ζ-potential response of PBMCs to 1.5-hour hyperosmotic challenge differentiated ME/CFS donors from healthy controls with 81.80% sensitivity and 85.70% specificity. This shows potential as a quantitative diagnostic biomarker.

The second study examined whether the electrophysiological properties of PBMCs could act as a correlate of protection to SARS-CoV-2. Cytoplasmic conductivity in unchallenged PBMCs was significantly reduced in donors who had received three SARS-CoV-2 vaccine doses compared with unmatched COVID-19 naïve donors. Stimulation with the receptor binding domain of the SARS-CoV-2 spike protein resulted in significant differences in normalised values of membrane conductance in third-dose vaccinated donors, from COVID-19 naïve and second-dose donors.

The third study investigated chondrocytes, which are used extensively in cell-based cartilage-repair therapies. Chondrocytes rapidly dedifferentiate and become fibroblastic during monolayer cell culture – decreasing the success of reimplantation surgery. Significant changes in chondrocyte electrophysiological properties were observed over time in culture, laying the foundations for the identification of an electrophysiological biomarker that correlates with chondrocytic phenotype, to improve re-implantation outcome.

These studies demonstrate novel applications of dielectrophoresis and ζ-potential analysis – to quantitatively diagnose ME/CFS, identify changes in PBMCs following COVID-exposure, and changes in chondrocyte electrophysiology during dedifferentiation.



Files and links (1)

PDF
Krista Clarke Final Thesis 11.28 MB
PDF Embargoed Access, Embargo ends: 01/07/2025

https://twitter.com/user/status/1807781027122643179

 

[Ravn]

Anyone been up to reading this?

Is study 1 relevant for understanding the OMF nano-needle results?

The three studies sound quite separate but does the author draw links between studies 1 (ME) and 3 (chondrocytes)? Because chondrocytes make extra-cellular matrix which has been discussed in a few threads lately as possibly relevant in ME

 

[SNT Gatchaman]

I think it's embargoed for a year.

 

[Ravn]

Ah, missed that date 2025

Did a quick search to see if some of the papers included in the thesis have been published separately as sometimes seems to happen but the only published paper the thesis author appears to have been involved with to date is one on some extremely technical aspects of dielectrophoresis. So we'll have to wait

 

[Ravn]

Is study 1 relevant for understanding the OMF nano-needle results?

Tentatively answering my own question: it might be relevant. Krista is working on the MEA/MERUK funded project that's following up on Davis' findings, see this thread

 

[Murph]

> 81.80% sensitivity and 85.70% specificity

That's decent. Although not the stark difference Stanford found:

Although the thesis talks about a 1.5 hour hyperosmotic challenge and the chart above shows the separation is not as strong at 1.5hour as it is a bit later.

 

[Murph]

I think it's embargoed for a year.

I just googled thesis embargos. They seem to imply something might be seeking publication or patenting. Which may be a hopeful sign.

 

[Sean]

That's decent. Although not the stark difference Stanford found:

Good clue, though. Well worth following up on.

 

[Simon M]

> 81.80% sensitivity and 85.70% specificity

That's decent. Although not the stark difference Stanford found:

Yes and – in fact, it’s not a very impressive results versus healthy controls. I hope it will be possible to refine this process. It was the scale of the separation in the nanoneedle results that was so striking.

 

[Sid]

Yes and – in fact, it’s not a very impressive results versus healthy controls. I hope it will be possible to refine this process. It was the scale of the separation in the nano Nigel results that Was so striking.

Hopefully they will submit something for publication so we can read more details about how they recruited controls. Sometimes healthy controls are not so healthy as in the case of the NIH study where the healthy controls were relatives of patients and in many cases had orthostatic intolerance.

 

[EndME]

> 81.80% sensitivity and 85.70% specificity

That's decent. Although not the stark difference Stanford found:

Although the thesis talks about a 1.5 hour hyperosmotic challenge and the chart above shows the separation is not as strong at 1.5hour as it is a bit later.

I don't think 81.80% sensitivity sounds bad. In my eyes it would probably be pretty close to what is achievable and considering the results of the intramural study and the problems of finding people with "true ME/CFS" is probably better than one can even expect. Anything much higher probably means you haven't identified a signal. I don't think specificity against controls from the general population matters too much either but you'd probably like to get some seperation from people with fatiguing illnesses and deconditioned people.

I think what will matter is whether there is a similar level of seperation as seen in the Stanford study where all controls looked similar but ME/CFS patients varied at different levels. In that case it might not matter if a few ME/CFS patients look similar to HCs, if the HCs all look somewhat similar. But if the results are all over the place and inconsistent things could be trickier.

More importantly will this tell us anything about pathology or might differences just be driven by being deconditioned, sample storage or something else? Has something like this been used to untangle pathology in a different illness?

 

[Sid]

https://meassociation.org.uk/2024/08/explaining-electrophysiological-properties-of-cells/

After my PhD, I am continuing to investigate these findings as a Research Fellow at the University of Surrey with the research team. I am very thankful to the ME Association and ME Research UK for funding this project, and to all the people who donate samples to the UK ME/CFS Biobank. Data gained from experiments using these samples has been invaluable.

We have come up with a way that separates ME/CFS donors from both healthy controls and multiple sclerosis disease controls using two relatively low-cost and commercially available devices, demonstrating promise towards identifying an adoptable diagnostic biomarker. The main research focus at the moment is to continue data collection to increase the validity of our findings and determine the sensitivity and specificity of the electrophysiological changes as a diagnostic biomarker in a larger donor cohort.

 

[SNT Gatchaman]

Embargo ends: 01/07/2025

So should be available in ~24 hours. Noted by Joan Crawford in this thread.

 

[forestglip]

Now available: Link | PDF

 

[ellesa]

In contrast with the significant increase in impedance unique to ME/CFS samples reported by Esfandyarpour et al. (2019), no clear trend could be seen in any dielectric parameter against time for donors in any cohort during incubation in hyperosmotic NaCl media in this small sample

A difference was observed between cohorts in the percentage change in ζ-potential (%) between 1.5-hour hyperosmotic NaCl incubation and 1.5-hour physiological incubation

 

[SNT Gatchaman]

Now we need to see bigger numbers, using fresh rather than frozen samples. And unlike the ubiquitous BPS "promising", this looks like it actually has potential

The initial plan for this study was to use fresh PBMC samples, by collecting whole blood from ME/CFS donors and performing same-day PBMC isolation and 3DEP experiments at the UK ME/CFS Biobank situated at the Royal Free Hospital in London. However, the commencement of these experiments planned for April 2020 were disrupted by the COVID-19 counter-measures which prohibited inter-county travel, restricted blood donations due to social distancing measures (further enforced by the vulnerability of ME/CFS patients), and limited laboratory access. To circumvent these complications and proceed with experiments, contingency measures included purchase of frozen PBMC aliquots.

Advantages of using fresh PBMC samples as opposed to frozen include eliminating potential changes in cellular electrical properties caused by damage to cell morphology during freezing (such as ice crystals puncturing cell membranes) or damage via the freezing medium – providing more accurate measurements of the hyperosmotic response of PBMCs in physiology.

Advantages of using the 3DEP and zetasizer machines over the nanoneedle array include they are easier to set up, low-cost, high-throughput, and readily available. In comparison the nanoneedle array is not used routinely, requires further instrumentational development to be deployed and is difficult. The biomarkers found in this study ensure absolutely no operator-bias or subjectivity, as they do not exclude outliers, technical repeats or in the case of the 3DEP, noisy DEP spectra. Furthermore, the biomarkers differentiate from MS disease controls, which has thus far been untested in impedance measurements using the nanoneedle assay. However, further experiments are required to increase the small sample size of this study to bring the statistical power to 0.95, and to recruit donors of other ethnicities and more male donors.

 

[Murph]

Here's another good chart from the thesis:



Figure 21| ζ-potential (mV) of PBMCs versus time during incubation in hyperosmotic NaCl medium in
|A| severe ME/CFS diagnosis donors (unchallenged black circles, NaCl blue triangles; n = 8)
|C| mild/moderate ME/CFS diagnosis (unchallenged black circles, NaCl red triangles; n = 9)
|E| healthy controls (unchallenged black circles, NaCl green triangles; n = 7)
|G| MS diagnosis (unchallenged black circles, NaCl purple triangles; n = 5). ζ-potential (mV) of PBMCs versus time during incubation
in hyperosmotic mannitol medium in |B| severe ME/CFS diagnosis donors (unchallenged black circles, mannitol blue triangles; n = 6) |D| mild/moderate ME/CFS diagnosis (unchallenged black circles, mannitol red triangles; n = 7)
|F| healthy controls (unchallenged black circles, mannitol green triangles; n = 5) |H| MS diagnosis (unchallenged black circles, mannitol purple triangles; n = 2). Mean datapoints plotted ± SEM. No statistical significance found using three-way ANOVA followed by Tukey’s multiple comparisons (*p<0.05).

It's not enough to create a conclusion that measuring 1.5 hours after a salt challenge is where you find a noticeable difference but it is enough to create a hypothesis worth testng!

and in case anyone is wondering, like I was before i hit google, this ζ is zeta.