blood tests – American ME and CFS Society

Replicated blood-based biomarkers for Myalgic Encephalomyelitis not explicable by inactivity

Abstract:

Myalgic Encephalomyelitis (ME; sometimes referred to as chronic fatigue syndrome) is a relatively common and female-biased disease of unknown pathogenesis that profoundly decreases patients’ health-related quality-of-life. ME diagnosis is hindered by the absence of robustly-defined and specific biomarkers that are easily measured from available sources such as blood, and unaffected by ME patients’ low level of physical activity.

Previous studies of blood biomarkers have not yielded replicated results, perhaps due to low study sample sizes (n<100). Here, we use UK Biobank (UKB) data for up to 1,455 ME cases and 131,303 population controls to discover hundreds of molecular and cellular blood traits that differ significantly between cases and controls. Importantly, 116 of these traits are replicated, as they are significant for both female and male cohorts.

Our analysis used semi-parametric efficient estimators, an initial Super Learner fit followed by a one-step correction, three types of mediators, and natural direct and indirect estimands, to decompose the average effect of ME status on molecular and cellular traits. Strikingly, these trait differences cannot be explained by ME cases’ restricted activity.

Of 3,237 traits considered, ME status had a significant effect on only one, via the “Duration of walk” (UKB field 874) mediator. By contrast, ME status had a significant direct effect on 290 traits (9%). As expected, these effects became more significant with increased stringency of case and control definition.

Significant female and male traits were indicative of chronic inflammation, insulin resistance and liver disease. Individually, significant effects on blood traits, however, were not sufficient to cleanly distinguish cases from controls. Nevertheless, their large number, lack of sex-bias, and strong significance, despite the ‘healthy volunteer’ selection bias of UKB participants, keep alive the future ambition of a blood-based biomarker panel for accurate ME diagnosis.

Source: Sjoerd V Beentjes, Julia Kaczmarczyk, Amanda Cassar, Gemma Louise Samms, Nima S Hejazi, Ava Khamseh, Chris P Ponting. Replicated blood-based biomarkers for Myalgic Encephalomyelitis not explicable by inactivity. medRxiv 2024.08.26.24312606; doi: https://doi.org/10.1101/2024.08.26.24312606 https://www.medrxiv.org/content/10.1101/2024.08.26.24312606v1 (Full text available as PDF file)

Role of pharmacological activity of autoantibodies in ME/CFS

Abstract:

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a condition characterised by extreme fatigue, memory impairment, pain and other symptoms that vary from patient to patient. It affects about 0.9% of the population and is often triggered by an acute viral or bacterial infection, such as Epstein-Barr virus. The underlying physiological and molecular basis of ME/CFS is unknown, and no effective treatments exist.

One proposed mechanism is that the blood flow is altered by autoantibodies against receptors involved in blood flow regulation. Antibodies are generated by the immune system to recognise intruders and under normal conditions, our immune system is trained not to attack our own tissues. However, during a severe infection, the immune system adopts an “all hands on deck” approach, which results in some of the newly-produced antibodies escaping quality control and targeting our own tissues, autoantibodies. Receptors regulation blood flow are located in walls of blood vessels and cause a blood vessel to dilate or contract as the demand for oxygen and nutrients to tissues such as the brain or muscles changes. Research has found increased levels of these autoantibodies in ME/CFS patients and initial trials removing these autoantibodies from the blood using a technique called immunoadsorption have shown improvement in symptoms.

In this project, we will test the hypothesis that autoantibodies can activate or inhibit the receptors responsible for the blood flow regulation, in a similar way medical drugs are used to regulate blood pressure.

We aim to profile serum samples from 325 ME/CFS patients and 130 healthy individuals to determine the presence of autoantibodies against all thirty receptors involved in blood pressure regulation. Importantly, we will study the ability of autoantibodies detected in each sample to activate or inhibit these receptors in order to test the hypothesis that the activity of these autoantibodies is a decisive factor in the disease.

If our hypothesis is correct, we will be able to develop an accurate blood test that may be able to detect ME/CFS earlier or to independently confirm the diagnosis. Ultimately, we hope that these results may also indicate a possible route for therapeutic intervention to counteract the effects of autoantibodies and alleviate the ME/CFS symptoms using a combination of already existing drugs, specific for each individual case.

Technical Summary:

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a condition of extreme tiredness and brain fog, often triggered by an acute infection. Its prevalence is ca 0.9% and here is no effective treatment. Competing theories for the root cause of ME/CFS include metabolic or redox homeostasis disruption, and presence of autoantibodies (AABs) against G protein coupled receptors (GPCRs) involved in regulation of blood flow.
Triggered by acute infection, autoimmunity is a result of reduced immuno-vigilance during severe infections, when an “all hands on deck” approach confers survival advantage. About 30% of ME/CFS patients show increased titre of autoantibodies against beta2-adrenoceptor and M3/4 muscarinic receptors controlling vasodilation/vasoconstriction, but this could become higher if all 30 receptors controlling blood flow would be taken into account.

In this project, we will test a hypothesis that the pharmacological activity of AABs against GPCRs is the key to their involvement in ME/CFS. Similar to medical drugs, AABs can be stimulatory (agonistic) or inhibitory (antagonistic) and induce a therapeutic or an undesired side effect.
We will profile 325 patient samples and 130 control plasma samples for AABs and their pharmacological activity using a state-of-the art GPCR drug screening pipeline we have established, against all 30 GPCRs involved in blood pressure regulation. We also have machine learning expertise that would allow us to interpret this extensive dataset, extract the most salient features. This will advance the understanding of the molecular basis of ME/CFS and could form the basis of a robust diagnostic blood test for ME/CFS. Ultimately, our findings may point in the direction of developing combination therapy using repurposed drugs to counteract the effects of autoantibodies and mitigate ME/CFS symptoms and stimulate the development of specific B-cell elimination strategy to cure ME/CFS.

Source: Lead Research Organisation: University of Nottingham, Department Name: School of Life Sciences. https://gtr.ukri.org/projects?ref=MR%2FY003667%2F1&pn=0&fetchSize=25&selectedSortableField=date&selectedSortOrder=ASC

Developing a blood cell-based diagnostic test for myalgic encephalomyelitis/chronic fatigue syndrome using peripheral blood mononuclear cells

Abstract:

A blood-based diagnostic test for myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and multiple sclerosis (MS) would be of great value in both conditions, facilitating more accurate and earlier diagnosis, helping with current treatment delivery, and supporting the development of new therapeutics.

Here we use Raman micro-spectroscopy to examine differences between the spectral profiles of blood cells of ME/CFS, MS and healthy controls.

We were able to discriminate the three groups using ensemble classification models with high levels of accuracy (91%) with the additional ability to distinguish mild, moderate, and severe ME/CFS patients from each other (84%).

To our knowledge, this is the first research using Raman micro-spectroscopy to discriminate specific subgroups of ME/CFS patients on the basis of their symptom severity. Specific Raman peaks linked with the different disease types with the potential in further investigations to provide insights into biological changes associated with the different conditions.

Source: Jiabao Xu, Tiffany Lodge,  Caroline Claire Kingdon, James W L Strong, John Maclennan, Eliana Lacerda, Slawomir Kujawski, Pawel Zalewski, Wei Huang, Karl J. Morten. Developing a blood cell-based diagnostic test for myalgic encephalomyelitis/chronic fatigue syndrome using peripheral blood mononuclear cells. medRxiv [Preprint] medRxiv 2023.03.18.23286575; doi: https://doi.org/10.1101/2023.03.18.23286575 https://www.medrxiv.org/content/10.1101/2023.03.18.23286575v1.full-text (Full text)

Autoimmune Gene Expression Proling of Fingerstick Whole Blood in Chronic Fatigue Syndrome

Abstract:

Background: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating condition that can lead to severe impairment of physical, psychological, cognitive, social, and occupational functions.

The cause of ME/CFS remains incompletely understood. There is no clinical diagnostic test for ME/CFS. Although many therapies have been used off-label to manage symptoms of ME/CFS, there are limited, if any, specific therapies or cure for ME/CFS.

In this study, we investigated the expression of genes specific to key immune functions, and viral infection status in ME/CFS patients with an aim of identifying biomarkers for characterization and/or treatment of the disease.

Methods: In 2021, one-hundred and sixty-six (166) patients diagnosed with ME/CFS and 83 healthy controls in the US participated in this study via a social media-based application (app). The patients and heathy volunteers consented to the study and provided self-collected finger-stick blood and first morning void urine samples from home.

RNA from the fingerstick blood was tested using DxTerity’s 51-gene autoimmune RNA expression panel (AIP). In addition, DNA from the same fingerstick blood sample was extracted to detect viral load of 4 known ME/CFS associated viruses (HHV6, HHV7, CMV and EBV) using a real-time PCR method.

Results: Among the 166 ME/CFS participants in the study, approximately half (49%) of the ME/CFS patients reported being house-bound or bedridden due to severe symptoms of the disease.

From the AIP testing, ME/CFS patients with severe, bedridden conditions displayed significant increases in gene expression of IKZF2, IKZF3, HSPA8, BACH2, ABCE1 and CD3D, as compared to 2 patients with mild to moderate disease conditions.

These six aforementioned genes were further upregulated in the 22 bedridden participants who suffer not only from ME/CFS but also from other autoimmune diseases.

These genes are involved in T cell, B cell and autoimmunity functions. Furthermore, IKZF3 (Aiolos) and IKZF2 (Helios), and BACH2 have been implicated in other autoimmune diseases such as systemic lupus erythematosus (SLE) and Rheumatoid Arthritis (RA).

Among the 240 participants tested with the viral assays, 9 samples showed positive results (including 1 EBV positive and 8 HHV6 positives).

Conclusions: Our study indicates that gene expression biomarkers may be used in identifying or differentiating subsets of ME/CFS patients having different levels of disease severity.

These gene targets may also represent opportunities for new therapeutic modalities for the treatment of ME/CFS. The use of social media engaged patient recruitment and at-home sample collection represents a novel approach for conducting clinical research which saves cost, time and eliminates travel for office visits.

Source: Zheng Wang, Michelle F. Waldman, Tara J. Basavanhally, Aviva R. Jacobs, et al. Autoimmune Gene Expression Proling of Fingerstick Whole Blood in Chronic Fatigue Syndrome. https://doi.org/10.21203/rs.3.rs-1942047/v1 (Full text)

Evaluating Routine Blood Tests According to Clinical Symptoms and Diagnostic Criteria in Individuals with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome

Abstract:

There is a lack of research regarding blood tests within individuals with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and between patients and healthy controls. We aimed to compare results of routine blood tests between patients and healthy controls. Data from 149 patients diagnosed with ME/CFS based on clinical and psychiatric evaluation as well as on the DePaul Symptom Questionnaire, and data from 264 healthy controls recruited from blood donors were compared. One-way ANCOVA was conducted to examine differences between ME/CFS patients and healthy controls, adjusting for age and gender.

Patients had higher sedimentation rate (mean difference: 1.38, 95% CI: 0.045 to 2.714), leukocytes (mean difference: 0.59, 95% CI: 0.248 to 0.932), lymphocytes (mean difference: 0.27, 95% CI: 0.145 to 0.395), neutrophils (mean difference: 0.34, 95% CI: 0.0 89 to 0.591), monocytes (mean difference: 0.34, 95% CI: 0.309 to 0.371), ferritin (mean difference: 28.13, 95% CI: -1.41 to 57.672), vitamin B12 (mean difference: 83.43, 95% CI: 62.89 to 124.211), calcium (mean difference: 0.02, 95% CI: -0.02 to 0.06), alanine transaminase (mean difference: 3.30, 95% CI: -1.37 to -7.971), low-density lipoproteins (mean difference: 0.45, 95% CI: 0.104 to 0.796), and total proteins (mean difference: 1.53, 95% CI: -0.945 to 4.005) than control subjects. The patients had lower potassium levels (mean difference: 0.11, 95% CI: 0.056 to 0.164), creatinine (mean difference: 2.60, 95% CI: 0.126 to 5.074) and creatine kinase (CK) (mean difference: 37.57, 95% CI: -0.282 to 75.422) compared to the healthy controls. Lower CK and creatinine levels may suggest muscle damage and metabolic abnormalities in ME/CFS patients.

Source: Baklund IH, Dammen T, Moum TÅ, Kristiansen W, Duarte DS, Castro-Marrero J, Helland IB, Strand EB. Evaluating Routine Blood Tests According to Clinical Symptoms and Diagnostic Criteria in Individuals with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome. J Clin Med. 2021 Jul 14;10(14):3105. doi: 10.3390/jcm10143105. PMID: 34300271. https://pubmed.ncbi.nlm.nih.gov/34300271/

Assessing cellular energy dysfunction in CFS/ME using a commercially available laboratory test

Abstract:

The mitochondrial energy score (MES) protocol, developed by the Myhill group, is marketed as a diagnostic test for chronic fatigue syndrome/Myalgic Encephalomyelitis (CFS/ME). This study assessed the reliability and reproducibility of the test, currently provided by private clinics, to assess its potential to be developed as an NHS accredited laboratory test.

We replicated the MES protocol using neutrophils and peripheral blood mononuclear cells (PBMCs) from CFS/ME patients (10) and healthy controls (13). The protocol was then repeated in PBMCs and neutrophils from healthy controls to investigate the effect of delayed sample processing time used by the Myhill group.

Experiments using the established protocol showed no differences between CFS/ME patients and healthy controls in any of the components of the MES (p ≥ 0.059). Delaying blood sample processing by 24 hours (well within the 72 hour time frame quoted by the Myhill group) significantly altered many of the parameters used to calculate the MES in both neutrophils and PBMCs. The MES test does not have the reliability and reproducibility required of a diagnostic test and therefore should not currently be offered as a diagnostic test for CFS/ME. The differences observed by the Myhill group may be down to differences in sample processing time between cohorts.

Source: Tomas C, Lodge TA, Potter M, Elson JL, Newton JL, Morten KJ. Assessing cellular energy dysfunction in CFS/ME using a commercially available laboratory test. Sci Rep. 2019 Aug 7;9(1):11464. doi: 10.1038/s41598-019-47966-z. https://www.ncbi.nlm.nih.gov/pubmed/31391529

Circulating extracellular vesicles as potential biomarkers in chronic fatigue syndrome/myalgic encephalomyelitis: an exploratory pilot study

Abstract:

Chronic Fatigue Syndrome (CFS), also known as Myalgic Encephalomyelitis (ME) is an acquired, complex and multisystem condition of unknown etiology, no established diagnostic lab tests and no universally FDA-approved drugs for treatment. CFS/ME is characterised by unexplicable disabling fatigue and is often also associated with numerous core symptoms. A growing body of evidence suggests that extracellular vesicles (EVs) play a role in cell-to-cell communication, and are involved in both physiological and pathological processes. To date, no data on EV biology in CFS/ME are as yet available.

The aim of this study was to isolate and characterise blood-derived EVs in CFS/ME. Blood samples were collected from 10 Spanish CFS/ME patients and 5 matched healthy controls (HCs), and EVs were isolated from the serum using a polymer-based method. Their protein cargo, size distribution and concentration were measured by Western blot and nanoparticle tracking analysis. Furthermore, EVs were detected using a lateral flow immunoassay based on biomarkers CD9 and CD63.

We found that the amount of EV-enriched fraction was significantly higher in CFS/ME subjects than in HCs (p = 0.007) and that EVs were significantly smaller in CFS/ME patients (p = 0.014). Circulating EVs could be an emerging tool for biomedical research in CFS/ME. These findings provide preliminary evidence that blood-derived EVs may distinguish CFS/ME patients from HCs. This will allow offer new opportunities and also may open a new door to identifying novel potential biomarkers and therapeutic approaches for the condition.

Source: Castro-Marrero J, Serrano-Pertierra E, Oliveira-Rodríguez M, Zaragozá MC, Martínez-Martínez A, Blanco-López MDC, Alegre J. Circulating extracellular vesicles as potential biomarkers in chronic fatigue syndrome/myalgic encephalomyelitis: an exploratory pilot study. J Extracell Vesicles. 2018 Mar 22;7(1):1453730. doi: 10.1080/20013078.2018.1453730. eCollection 2018. https://www.ncbi.nlm.nih.gov/pubmed/29696075

NMR metabolic profiling of serum identifies amino acid disturbances in chronic fatigue syndrome

Abstract:

Chronic fatigue syndrome (CFS) is a debilitating multisystem disorder characterised by long-term fatigue with a variety of other symptoms including cognitive dysfunction, unrefreshing sleep, muscle pain, and post-exertional malaise. It is a poorly understood condition that occurs in ~5 in every 1000 individuals. We present here a preliminary study on the analysis of blood samples from 11 CFS and 10 control subjects through NMR metabolic profiling.

Identified metabolites that were found to be significantly altered between the groups were subjected to correlation analysis to potentially elucidate disturbed metabolic pathways. Our results showed a significant reduction of glutamine (P=0.002) and ornithine (P<0.05) in the blood of the CFS samples. Correlation analysis of glutamine and ornithine with other metabolites in the CFS sera showed relationships with glucogenic amino acids and metabolites that participate in the urea cycle. This indicates a possible disturbance to amino acid and nitrogen metabolism. It would be beneficial to identify any potential biomarkers of CFS for accurate diagnosis of the disorder.

Source: Armstrong CW, McGregor NR, Sheedy JR, Buttfield I, Butt HL, Gooley PR. NMR metabolic profiling of serum identifies amino acid disturbances in chronic fatigue syndrome. Clin Chim Acta. 2012 Oct 9;413(19-20):1525-31. doi: 10.1016/j.cca.2012.06.022. Epub 2012 Jun 21. https://www.ncbi.nlm.nih.gov/pubmed/22728138

Spectroscopic diagnosis of chronic fatigue syndrome by visible and near-infrared spectroscopy in serum samples

Abstract:

To investigate visible and near-infrared (Vis-NIR) spectroscopy enabling chronic fatigue syndrome (CFS) diagnosis, we subjected sera from CFS patients as well as healthy donors to Vis-NIR spectroscopy. Vis-NIR spectra in the 600-1100 nm region for sera from 77 CFS patients and 71 healthy donors were subjected to principal component analysis (PCA) and soft independent modeling of class analogy (SIMCA) to develop multivariate models to discriminate between CFS patients and healthy donors. The model was further assessed by the prediction of 99 masked other determinations (54 in the healthy group and 45 in the CFS patient group).

The PCA model predicted successful discrimination of the masked samples. The SIMCA model predicted 54 of 54 (100%) healthy donors and 42 of 45 (93.3%) CFS patients of Vis-NIR spectra from masked serum samples correctly. These results suggest that Vis-NIR spectroscopy for sera combined with chemometrics analysis could provide a promising tool to objectively diagnose CFS.

Source: Sakudo A, Kuratsune H, Kobayashi T, Tajima S, Watanabe Y, Ikuta K. Spectroscopic diagnosis of chronic fatigue syndrome by visible and near-infrared spectroscopy in serum samples. Biochem Biophys Res Commun. 2006 Jul 14;345(4):1513-6. Epub 2006 May 22. https://www.ncbi.nlm.nih.gov/pubmed/16730652

Chronic fatigue syndrome and/or fibromyalgia as a variation of antiphospholipid antibody syndrome: an explanatory model and approach to laboratory diagnosis

Abstract:

Chronic Fatigue and/or Fibromyalgia have long been diseases without definition. An explanatory model of coagulation activation has been demonstrated through use of the ISAC panel of five tests, including, Fibrinogen, Prothrombin Fragment 1+2, Thrombin/ AntiThrombin Complexes, Soluble Fibrin Monomer, and Platelet Activation by flow cytometry. These tests show low level coagulation activation from immunoglobulins (Igs) as demonstrated by Anti-B2GPI antibodies, which allows classification of these diseases as a type of antiphospholipid antibody syndrome. The ISAC panel allows testing for diagnosis as well as monitoring for anticoagulation protocols in these patients.

Source: Berg D, Berg LH, Couvaras J, Harrison H. Chronic fatigue syndrome and/or fibromyalgia as a variation of antiphospholipid antibody syndrome: an explanatory model and approach to laboratory diagnosis. Blood Coagul Fibrinolysis. 1999 Oct;10(7):435-8. http://www.ncbi.nlm.nih.gov/pubmed/10695770