Erythrocyte Deformability As a Potential Biomarker for Chronic Fatigue Syndrome (Davis, 2018)

[Diwi9]

Dr. Ron Davis has been speaking about blood cell deformability in ME/CFS for many months and has finally published on his research: http://www.bloodjournal.org/content/132/Suppl_1/4874?sso-checked=true

Abstract
Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is arguably the last major disease we know almost nothing about. It is a multi-systemic illness of unknown etiology affecting millions of individuals worldwide, with the capacity to persist for several years. ME/CFS is characterized by disabling fatigue of at least 6 months, accompanied serious fatigue and musculoskeletal pain, in addition to impaired short-term memory or concentration, and unrefreshing sleep or extended post-exertional. While the etiology of the disease is still debated, evidence suggest oxidative damage to immune and hematological systems as one of the pathophysiological mechanisms of the disease. Erythrocytes are potent scavengers of oxidative stress, and their shape changes appreciably in response to oxidative stress and certain inflammatory conditions including obesity and diabetes. The shape of erythrocytes change from biconcave discoid to an ellipsoid due shear flow in microcapillaries that provides a larger specific surface area-to-volume ratio for optimal microvascular perfusion and tissue oxygenation establishing the importance not only of total hematocrit but also of the capacity for large deformations in physiology. Clinically, ME/CFS patients show normal arterial oxygen saturation but nothing much is known about microvascular perfusion. In this work, we tested the hypothesis that the erythrocyte deformability in ME/CFS is adversely affected, using a combination of biophysical and biochemical techniques.

We tested the deformability of RBCs using a high-throughput microfluidic device which mimics blood flow through microcapillaries. We perfused RBCs (suspension in plasma) from ME/CFS patients and from age and sex matched healthy controls (n=9 pairs of donors) through a high-throughput microfluidic platform of 5µm width and 3-5 µm height. We recorded the movement of the cells at high speed (4000 fps), followed by image analysis to assess the following parameters: entry time (time required by the cells to completely enter the test channels), average transit velocity (velocity of the cells inside the test channels) and elongation index (ratio of the major diameter before and after deformation in the test channel). We observed that RBCs from ME/CFS patients had higher entry time (~12%, p<0.0001), lower average transit velocity (~17%, p<0.0001) and lower elongation index (~14%, p<0.0001) as compared to RBCs from healthy controls. Taken together, this data shows that RBCs from ME/CFS patients have reduced deformability. To corroborate our findings, we also measured the erythrocyte sedimentation rate (ESR) for these donors which show that the RBCs from ME/CFS patients had lower (~40%, p<0.01) sedimentation rates.

To understand the basis for differences in deformability, we investigated the changes in the fluidity of the membrane using a lateral diffusion assay using pyrenedecanoic acid (PDA), and observed that RBCs from ME/CFS patients have lower membrane fluidity (~30%, p<0.01). Apart from the fluidity, Zeta potential measurements showed that ME/CFS patients had lower net negative surface charge on the RBC plasma membrane (~18%, p<0.0001). Higher levels of reactive oxygen species (ROS) in RBCs from ME/CFS patients (~30%, p<0.008) were also observed, as compared to healthy controls. Using scanning electron microscopy (SEM), we also observed changes in RBC morphology between ME/CFS patients and healthy controls (presence of different morphological subclasses like biconcave disc, leptocyte, acanthocyte and burr cells; area and aspect ratio; levels of RBC aggregation). Despite these changes in RBC physiology, the hemoglobin levels remained comparable between healthy donors and ME/CFS patients. Finally, preliminary studies show that RBCs from recovering ME/CFS patients do not show such differences in cellular physiology, suggesting a connection between RBC deformability and disease severity.

Taken together, our data demonstrates that the significant decrease in deformability of RBCs from ME/CFS patients may have origins in oxidative stress, and suggests that altered microvascular perfusion can be a possible cause for ME/CFS symptoms. Our data also suggests that RBC deformability may serve as a potential biomarker for ME/CFS, albeit further studies are necessary for non-specific classification of the disease.

 

[Countrygirl]

Isn't this what we were reading about 30 years ago? Was it Dr Learner's work from Australia?

I am glad Dr Davies has published at last, but it makes me weep that we have not moved further forward in all that time.

 

[Countrygirl]

This is a useful dummy's guide to the subject.

https://www.omf.ngo/2018/04/04/rbc-shape-rbc-deformability/

 

[Murph]

super pleased this is out.

They associate the membrane problems with reactive oxygen species. I'd been thinking more inherent membrane structure problems so that's an interesting new perspective.

Hopefully they can scale up this nine patient study enough to do some subgroup analysis. (it already implies they have tested on a 'recovering' person and found their blood cells plenty deformable, so that's a kind of subgroup.)

I'd be very interested in longitudinal analyses, especially around PEM. Is this something that changes day to day?

Lastly I note their p-values are very low for a nine-person, nine-control study and I conclude their sample size is based on counting actual red blood cells, not just patients! Is that appropriate? idk.

 

[halcyon]

Isn't this what we were reading about 30 years ago? Was it Dr Learner's work from Australia?

I am glad Dr Davies has published at last, but it makes me weep that we have not moved further forward in all that time.

It was Les O. Simpson from New Zealand. He was working on this in the 1980s.

 

[Diwi9]

@Murph - My hope is that publication will help Ron receive NIH grant funding that can facilitate a large-scale study

 

[pattismith]

@alex3619
@Murph

It may be that low deformability of red blood cells could be linked to low omega 3 in CFS/ME patients.


At less, omega 3 supplementation is likely to improve this problem, because DHA was shown to greatly ameliorate deformability in some cases:


Dietary supplementation with docosahexanoic acid (DHA) increases red blood cell membrane flexibility in mice with sickle cell disease


Abstract
Humans and mice with sickle cell disease (SCD) have rigid red blood cells (RBCs). Omega-3 fatty acids, such as docosahexanoic acid (DHA), may influence RBC deformability via incorporation into the RBC membrane. In this study, sickle cell (SS) mice were fed natural ingredient rodent diets supplemented with 3% DHA (DHA diet) or a control diet matched in total fat (CTRL diet). After 8 weeks of feeding, we examined the RBCs for: 1) stiffness, as measured by atomic force microscopy; 2) deformability, as measured by ektacytometry; and 3) percent irreversibly sickled RBCs on peripheral blood smears. Using atomic force microscopy, it is found that stiffness is increased and deformability decreased in RBCs from SS mice fed CTRL diet compared to wild-type mice.
In contrast, RBCs from SS mice fed DHA diet had markedly decreased stiffness and increased deformability compared to RBCs from SS mice fed CTRL diet.
Furthermore, examination of peripheral blood smears revealed less irreversibly sickled RBCs in SS mice fed DHA diet as compared to CTRL diet. In summary, our findings indicate that DHA supplementation improves RBC flexibility and reduces irreversibly sickled cells by 40% in SS mice. These results point to potential therapeutic benefits of dietary omega-3 fatty acids in SCD.

 

[Murph]

Here's an interesting effect on red blood cells that they were unable to detect 5 minutes after exercise, but could detect 24 hours later.


Effects of swimming exercise on red blood cell rheology in trained and untrained rats.
Yalcin, Ozlem, Melek Bor-Kucukatay, Umit K. Sen- turk, and Oguz K. Baskurt.
J Appl Physiol 88: 2074–2080, 2000.—

Red blood cell (RBC) mechanical properties were investigated after swimming exercise in trained and untrained rats. A group of rats was trained for 6 wk (60 min swimming, daily), and another group was kept sedentary. Blood samples were obtained either within 5 min or 24 h after 60 min swimming in both groups. In the untrained rats, the RBC aggregation index decreased to 2.60 0.4 immediately after exercise from a control value of 6.73 0.18 (P 0.01), whereas it increased to 13.13 0.66 after 24 h (P 0.01). RBC transit time through 5-μm pores increased to 3.53 0.16 ms within 5 min after the exercise from a control value of 2.19 0.07 ms (P 0.005). A very significant enhancement (166%) in RBC lipid peroxidation was detected only after 24 h.

If you're looking at that and thinking about PEM, you're thinking what I'm thinking. Lipid peroxidation is about oxidisation, where free radicals attack cell membranes.

I am seriously taken with this whole redox/red blood cell deformability hypothesis.

 

[Belbyr]

Isn't this what we were reading about 30 years ago? Was it Dr Learner's work from Australia?

I am glad Dr Davies has published at last, but it makes me weep that we have not moved further forward in all that time.

Well, you can also add that there are hypothesis of the kynurenine/tryptophan 'metabolic trap' dating back to as early as 2011. I think it is the technology and will power that have emboldened the researchers recently. Just like I have been watching Nancy Klimas... They had the computer modeling of ME/CFS and GWS worked out back around 2011 as well. It was the cell and mouse model tests that emboldened them just recently to put the effort and money into the first human trials that are underway right now.

 

[nandixon]

Here's an interesting effect on red blood cells that they were unable to detect 5 minutes after exercise, but could detect 24 hours later.

Effects of swimming exercise on red blood cell rheology in trained and untrained rats.
Yalcin, Ozlem, Melek Bor-Kucukatay, Umit K. Sen- turk, and Oguz K. Baskurt.
J Appl Physiol 88: 2074–2080, 2000.—
...
A very significant enhancement (166%) in RBC lipid peroxidation was detected only after 24 h.

If you're looking at that and thinking about PEM, you're thinking what I'm thinking. Lipid peroxidation is about oxidisation, where free radicals attack cell membranes.

I am seriously taken with this whole redox/red blood cell deformability hypothesis.

The same authors then did a follow-up study published one year later (in 2001) and found that simply supplementing vitamin C and vitamin E completely prevented the lipid peroxidation and reduced rbc deformability issue that otherwise happened 24 hours later in the untrained (“Sed”) rats:

Whereas exhaustion lead to decreased erythrocyte deformability (increased TT) and increased plasma hemoglobin concentration levels, antioxidant treatment before exhaustion gave protection from these harmful effects in Sed rats.
https://www.physiology.org/doi/full/10.1152/jappl.2001.91.5.1999

And that's the problem with thinking that reduced red blood cell deformability is a key issue in ME/CFS (as I mentioned previously on the parallel thread here), because the oxidative stress/lipid peroxidation that's causing it is easily corrected with the usual array of antioxidants that most people with ME/CFS should already be taking, i.e., vitamin E, NAC, selenium, etc. - whatever amounts and combinations a person can tolerate. (I can't use alpha-lipoic acid for instance.)

Are you not already taking those? If you are, then it's very likely you've already corrected any potential red blood cell deformability issue. A lipid peroxidation test would let you know. If lipid peroxidation is normal then rbc deformability is going to be normal as well, absent some other disease process going on.

Now, if Ron Davis's study showed reduced rbc deformability while simultaneously showing normal lipid peroxidation then that would be a remarkable finding. And a problematic one as well because it would mean the patient(s) has another disease going on instead of or in addition to ME/CFS - one that causes an inherent structural problem in the rbc membrane.

 

[Murph]

Are you not already taking those?

I'm about to start taking those!