Bioimpedance spectroscopy characterization of osmotic stress processes in MECFS blood samples, 2023 Fernandez et al

[Jaybee00]

Now published - details at this post
Preprint detail below
***

https://www.authorea.com/users/6382...chronic-fatigue-syndrome-me-cfs-blood-samples


Bioimpedance spectroscopy characterization of osmotic stress processes in Myalgic Encephalomyelitis / Chronic Fatigue Syndrome (ME-CFS) blood samples

Abstract
Myalgic Encephalomyelitis / Chronic Fatigue Syndrome (ME/ CFS) is a disabling, chronic, multi-system and complex disease. Currently, there are no specific laboratory tests to directly [diagnose ME/CFS](https://www.cdc.gov/me-cfs/symptoms-diagnosis/diagnosis.html). In this work we study the use of impedance spectroscopy as a potential technique for the diagnosis of this disease. A specific device for the electrical characterization of peripheral blood mononuclear cells was designed and implemented. Impedance spectroscopy measurements in the range from 1 Hz to 500 MHz were made after osmotic stress of the samples with sodium chloride solution 1M. The evolution in time after the osmotic stress at two specific frequencies (1.36 kHz and 154 kHz) was analysed. The device showed its sensitivity to the presence of cells and the evolution of the osmotic process. Higher values of impedance were measured for 1.36 kHz in ME/CFS patients compared to control samples. Results help to further understand the relation of bioimpedance measurements with ME/CFS samples physical properties and osmotic processes.




Conclusions
In this work we study the use of impedance spectroscopy as a potential technique for the diagnosis of Myalgic Encephalomyelitis / Chronic Fatigue Syndrome (ME/ CFS). We have analysed peripheral blood mononuclear cells from four chronic fatigue patients and four healthy controls, after an osmotic stress of the samples with NaCl solution 1M.

A specific device for impedance measurement was designed and implemented, consisting on two interdigitated electrodes placed on a Petri dish, connected to an impedance analyser, obtaining measurements in the range from 1 Hz to 500 MHz. The device showed its good sensitivity to the presence of cells and the addition of NaCl to stress the cells, being an affordable option for a potential diagnostic solution in medical laboratories.
Real part and imaginary part of impedance were analysed for the whole duration of the experiment at two specific frequencies. At 1.36 kHz, higher values of impedance (both in the real part and the imaginary part, in absolute values) were measured in ME/CFS patients, in comparison with control individuals. As this frequency is located in the alpha dispersion region, the difference of impedance may be related to the effect of extra-cellular ions. At 154 kHz, no significant difference has been found between patient samples and control samples.

 

[Hutan]

Figure 4 shows the results of the measured impedance at 1.36 kHz for the different patient and control samples, at different times of the experiments described in the Materials and Methods section. We can observe for all frequencies (figure 3A) a decrease in the real part of the measured impedance at 30 minutes, as expected after the addition of NaCl solution. The bioimpedance values then start to rise, and stabilize at the end of the experiment. In opposition with [8], no significant increase in the impedance measurements have been found for the patient samples after one hour of measurements, at 1.36 kHz.
Figure 4C and 4D show the mean values of the real part and imaginary part of the measured bioimpedance at 1.36 kHz. Higher values of impedance (both in the real part and the imaginary part, in absolute values) are measured in ME/CFS patients, in comparison with control individuals, as can be seen in figure 4C and 4D, throughout the whole duration of the experiment.

I'm well out of my comfort zone on this one, and it looks like a small homemade experiment with some charts that aren't ideal. But, well done to the authors for getting on and doing something and writing it up.

It looks to me as though the impedance of a solution with a standardised number of PBMC cells differs for ME/CFS and healthy samples at a frequency of 1.36 kHz. And that adding the salt at 30 minutes doesn't change that - there are still consistent differences.



They don't replicate the findings of the team from Ron Davis's lab. But perhaps the higher impedance finding in the ME/CFS samples is interesting. The authors seem to be able to tell from the frequency that the impedance difference is due to extra-cellular ions. They plan to do more experiments.

Here's the picture of the sensor and the circuit in the Petri dish of cell solution.

 

[chillier]

It's a very small experiment with 4 controls and 4 'chronic fatigue' patients with no mention of diagnosis criteria or age and sex matching etc. Plots aren't great and the panels aren't labelled which makes it a bit hard to read. As well as the barplots @Hutan posted they show the data as a scatter plot and it looks like the controls and patients overlap but it's really awkward to read.

They mention in the abstract they treat the cells with 1M NaCl which I was surprised by because this concentration would completely destroy the cells. Doing the maths though it looks like they are using the same salt stress set up as in Ron's nanoneedle - 200mM NaCl:

Once we had the cellular pellet, it was resuspended in 1 mL of PBS and a cell count was carried out in a Neubauer Improved camera. The concentration of cells was adjusted to 200,000/mL in 2 mL of the plasm previously obtained.
The cell plus plasm mix was placed in a Petri plate with the measurement device, and the bioimpedance was measured with the MFIA instrument, from Zurich Instruments, 20 minutes to obtain the basal data. After those 20 minutes, 120 uL of NaCl 1M was added to the mix to stress the cells and the bioimpedance was measured 90 additional minutes.

cells are resuspended in 2mls PBS which contains 0.9% NaCl (0.9grams in 100ml). So (0.9*10)(g/L) / 58.44(g/mol) = 0.15M = 150mM which is an isotonic solution which the cells will be happy in.

They add 120uL of 1M NaCl to the 2mls so that's a dilution of 120uL in 2120uL -> 0.057 * 1M = 0.057M NaCl aka 57mM.

So the final concentration is 150mM of the buffer + 57mM from the added NaCl to 207mM which is basically the same as that used in Ron's nanoneedle.

In Ron's nanoneedle they use a frequency of 15Khz, in this paper they try a range of frequencies but land on using 1.36KHz and 154KHz. Different frequencies interact with the cells and media in different ways and I won't go into that too much but at a low frequency the membranes are physically blocking current and at higher frequencies the current can pass through the membrane and you're measuring other things. It's unclear exactly how to map the frequencies used onto what biological properties you're measuring. They claim in this paper that at 1.36KHz the membrane is basically blocking current flow so they are instead measuring ions in the solution surrounding the cells, and that at 154Khz you can measure internal properties of the cell/ polarisation of the membrane itself.

They use the same concentration of cells 200,000 cells/ml in both this paper and Ron's nanoneedle though of course with different total volumes 50uL (nanoneedle) versus 2ml in this paper.

The geometry of this experimental set up is very different to Ron's nanoneedle. Here's a side on view of how I imagine the cells in the petri dish:


Cells are in green (~30micron diameter). Red and blue are the anodes and cathodes sitting flush on the bottom of the petri dish. They are either 0.2mm apart from each other or 0.2mm in diameter the paper doesn't make it totally clear - let's assume 0.2mm apart from each other. That's the width of approximately 7 cells assuming a cell diameter of about 30microns. The cells are also unlikely to be that closely positioned to the electrodes as they'll be floating in the media. Cells will sink over the course of hours but it looks like they get on with the readings immediately. Could mean that the impedance of the cells themselves aren't really being measured as they're too far away - not sure.

This is how I think the geometry of Ron's nanoneedle goes:


Cells are squeezed into a channel only slightly larger than their diameter and so the cells are (I imagine) squeezed directly against the electrode (and possibly measuring membrane impedance) or sitting very nearby (and measuring ion/molecule impedance of the medium surrounding the cells, possibly with a higher local concentration due to the proximity of the cells). Could also be possibly measuring internal cytoplasmic properties of the cell depending on the capacitative properties of the membrane at the frequency used - in other words the current is able to pass directly through the membrane.

The distance between the electrodes is ~2microns, 200x smaller than that above. Presumably this difference in distance that has to be covered affects the readings somehow but I don't know exactly. In neither experimental set up are the anode and cathode held straightforwardly on opposing sides of a cell, so the actual path the current is taking isn't totally clear.

 

[Creekside]

At this point it's not really important just what they're measuring. What's important is finding a reliable difference between PWME and people without ME. Since this is only 4 samples, without mentioning the criteria, I think it's a proof of concept for a deeper study.

 

[Jonathan Edwards]

I think it's a proof of concept for a deeper study.

Proof of concept is when you have actually proven the concept - i.e. got a definitive result!

 

[Creekside]

I meant an attempt at proof of "being worth continuing". It got an interesting result, but was that just a glitch, or something to follow up on. If it showed no interesting results, well, it was worth trying, but not worth continuing.

 

[Simon M]

@chillier thanks for the very helpful analysis and explanation (and for confirming that it is the same salt concentration as used in Ron Davis' experiment).

It looks interesting, but they don’t replicate the nanoneedle findings. However, this experiment seems to have very significant limitations:

– Only four controls and four “chronic fatigue“patients.
– A “home-made“ system @Hutan, rather than an existing one that had been developed to get the best results.
– Strange “geometry“, with cells apparently floating above the electrodes. I’ve only seen a couple of other systems for measuring impedance, but they seem to rely on cells as a layer at the bottom.
– This new study also uses a different frequency to the Ron Davis experiment, though maybe they did try the same frequency and it made no difference.

So I think this is hard to interpret. I’m glad to hear they are planning further experiments.

 

[Dolphin]

Now published:
http://thesciencepublishers.com/biomed_lett/v9i2abstract6.html

https://twitter.com/user/status/1713015296980574581

 

[Hutan]

Bioimpedance spectroscopy characterization of Myalgic Encephalomyelitis/ Chronic Fatigue Syndrome (ME/CFS) peripheral blood mononuclear cells
Sara Martínez Rodríguez1, Alberto Olmo Fernández 2,3 *, Daniel Martín Fernández 3,4, Isabel Martín-Garrido 1,5


https://idus.us.es/bitstream/handle/11441/149595/1/Articulo.pdf?sequence=1

Abstract

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a disabling and chronic disease, importantly related to the current COVID-19 pandemic. Currently, there are no specific laboratory tests to directly diagnose ME/CFS. In this work, the use of impedance spectroscopy is studied as a potential technique for the diagnosis of ME/CFS. A specific device for the electrical characterization of peripheral blood mononuclear cells was designed and implemented. Impedance spectroscopy measurements in the range from 1 Hz to 500 MHz were carried out after the osmotic stress of the samples with sodium chloride solution at 1M concentration. The evolution in time after the osmotic stress at two specific frequencies (1.36 kHz and 154 kHz) was analyzed.

The device showed its sensitivity to the presence of cells and the evolution of the osmotic processes. Higher values of impedance (around 15% for both the real and imaginary part) were measured at 1.36 kHz in ME/CFS patients compared to control samples. No significant difference was found between patient samples and control samples at 154 kHz. Results help to further understand the diagnosis of ME/CFS patients and the relation of their blood samples with bioimpedance measurements.