Tag: peripheral blood mononuclear cells

Using Single-Cell Raman Microspectroscopy to Profile Human Peripheral Blood Mononuclear Cells

Abstract:

A reliable, validated test would enhance our ability to treat and research chronic conditions. Early and accurate diagnosis would provide an entry point into clinical care, give access to benefits, remove the stigma associated with these conditions, and importantly, provide researchers with a fundamental tool they require to study these heterogeneous disorders.

In this chapter, we describe how Raman microspectroscopy can be utilised to study the biology of peripheral blood mononuclear cells (PBMCs) isolated from human blood samples. Using machine learning approaches, the data generated can be used to attempt to separate different patient and control groups, subgroups within a patient cohort, and identify differences in intracellular metabolites which may provide clues about disease mechanisms.

Source: Gan E, Stoker M, Guo E, Morten KJ, Xu J. Using Single-Cell Raman Microspectroscopy to Profile Human Peripheral Blood Mononuclear Cells. Methods Mol Biol. 2025;2920:29-37. doi: 10.1007/978-1-0716-4498-0_3. PMID: 40372676. https://link.springer.com/protocol/10.1007/978-1-0716-4498-0_3

Deep Immunophenotyping in ME/CFS Using Spectral Flow Cytometry

Abstract:

Immune dysfunction is reported to play a significant role in the etiology of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). To gain an understanding of the underlying immune abnormalities associated with this complex condition, a comprehensive approach for characterizing immune cell subsets and their inferred functional states is essential.

We developed a high-dimensional flow cytometry method that enables detailed immunophenotyping of peripheral blood mononuclear cells (PBMCs) from ME/CFS patients. By simultaneously measuring over 40 markers on individual cells within one sample, this approach provides a comprehensive assessment of immune cell subsets, incorporating effector or functional states, to enable assessment of their potential roles in disease pathogenesis.

Source: Gibson A, Chometon TQ, Damani T, Brooks AES. Deep Immunophenotyping in ME/CFS Using Spectral Flow Cytometry. Methods Mol Biol. 2025;2920:59-82. doi: 10.1007/978-1-0716-4498-0_5. PMID: 40372678. https://link.springer.com/protocol/10.1007/978-1-0716-4498-0_5

Analysis of Transient Receptor Potential Ion Channels in ME/CFS

Abstract:

This chapter provides a comprehensive overview of methodologies currently employed to study ion channels, particularly transient receptor potential melastatin 3 (TRPM3) in the context of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Sample preparation involves the collection of whole blood, separation of peripheral blood mononuclear cells (PBMCs) via density gradient centrifugation, and isolation of natural killer (NK) cells.

Protein expression analysis utilizes flow cytometry, liquid chromatography-mass spectrometry (LC-MS), western blotting, and immunofluorescence techniques. Functional analysis focuses on calcium imaging and electrophysiology techniques to investigate ion channel responses to pharmacological stimuli.

The authors highlight that some experimental protocols included within this chapter require specialized training and equipment. In order to replicate these protocols extended training is advised, specifically when attempting electrophysiology experimentation. The use of advanced techniques for detailed analysis provides insights into ion channel function and potential implications in the pathomechanism of ME/CFS offering avenues for further research and therapeutic exploration.

Source: Eaton-Fitch N, Muraki K, Sasso EM, Magawa C, Marshall-Gradisnik S. Analysis of Transient Receptor Potential Ion Channels in ME/CFS. Methods Mol Biol. 2025;2920:83-99. doi: 10.1007/978-1-0716-4498-0_6. PMID: 40372679. https://link.springer.com/protocol/10.1007/978-1-0716-4498-0_6

Stress-Induced Changes in Immune Signatures in ME/CFS Patients Determined by Transcriptome Analysis

Abstract:

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic, complex multi-organ illness characterized by unexplained debilitating fatigue and post-exertional malaise (PEM). We evaluated transcriptomic changes in peripheral blood mononuclear cells (PBMC) of ME/CFS patients undergoing an exercise challenge and explored the transcriptomic response to exercise and recovery in PBMC of ME/CFS patients, as compared to healthy controls using RNA sequencing technology. As transcriptomic changes in ME/CFS patients are still in the phase of discovery, analysis of data has to be stringent, and the most important results have to be validated by a different technology, such as real-time PCR or NanoString.

Source: Gamer J, Van Booven D, Zarnowski O, Perez M, Frank J, Pangeni RP, Collado F, Klimas NG, Oltra E, Nathanson L. Stress-Induced Changes in Immune Signatures in ME/CFS Patients Determined by Transcriptome Analysis. Methods Mol Biol. 2025;2920:103-112. doi: 10.1007/978-1-0716-4498-0_7. PMID: 40372680. https://link.springer.com/protocol/10.1007/978-1-0716-4498-0_7

Quantitative Proteomics on Immune Cells of ME/CFS Patients Using SWATH-MS

Abstract:

Proteomics is one of the “omics” disciplines that has provided molecular insights into the pathophysiology of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Here we describe a complete SWATH-MS workflow for the quantitative profiling of proteins extracted from peripheral mononuclear blood cells to investigate proteomic alterations in ME/CFS. This workflow covers all steps of sample preparation, data acquisition, and data analysis.

We describe the process of generating a comprehensive spectral library from a pre-fractionated peptide reference sample followed by the acquisition of DIA data sets of individual samples using a 5600+ TripleTOF mass spectrometer. Examples of both library-based and library-free data analysis pipelines are presented based on the PeakView/MarkerView software package (commercial) and DIA-NN (free) software respectively.

Source: Kumar A, Peppercorn K, Kleffmann T. Quantitative Proteomics on Immune Cells of ME/CFS Patients Using SWATH-MS. Methods Mol Biol. 2025;2920:113-140. doi: 10.1007/978-1-0716-4498-0_8. PMID: 40372681. https://link.springer.com/protocol/10.1007/978-1-0716-4498-0_8

Measurement of Genetic Variations in ME/CFS Patients in the IDO2 Gene Encoding an Enzyme Metabolizing Tryptophan

Abstract:

Genetic variations in the indoleamine 2,3-dioxygenase (IDO2) gene that are commonly found in the general population have been assessed for their frequency in myalgic encephalomyelitis/chronic fatigue (ME/CFS) patients compared with healthy controls. They have potential for being genetic variations that lead to susceptibility to developing ME/CFS following exposure to a triggering stressor like a viral infection or other major stress events.

The IDO2 gene encodes an enzyme that is involved in the tryptophan-kynurenine pathway (TKP), and is activated if there are excessive amounts of tryptophan to prevent excessive serotonin production. The TKP pathway through production of NADH is involved in regulating the immune system and likely plays an important role in ME/CFS.

A simple method was developed to evaluate the 5 commonly occurring mutations in this gene in ME/CFS patients and to determine if one or more were present at higher frequency than in healthy controls. This might indicate a susceptibility factor for developing ME/CFS. In this chapter we describe the techniques used to isolate peripheral blood mononuclear cells (PBMCs), extract the DNA, and then do touchdown PCR and DNA sequencing for the analysis.

Source: Edgar CD, Blair A, Tate WP. Measurement of Genetic Variations in ME/CFS Patients in the IDO2 Gene Encoding an Enzyme Metabolizing Tryptophan. Methods Mol Biol. 2025;2920:247-256. doi: 10.1007/978-1-0716-4498-0_14. PMID: 40372687. https://link.springer.com/protocol/10.1007/978-1-0716-4498-0_14

Persistence of SARS-CoV-2 in Platelets and Megakaryocytes in Long COVID

Abstract:

Background: We have shown that acute COVID-19 pathophysiology is profoundly altered by infection of lung megakaryocytes (MKs) and platelets by SARS‑CoV‑2 (Zhu et al, 2022). A significant proportion of COVID-19 patients have symptoms persisting for > 3 months after initial infection with SARS-CoV-2, referred to as Long COVID or Post-acute Sequelae of SARS-CoV-2 (PASC) patients. Persistent or re-emerging symptoms are varied, with a predominance of asthenia, neuro-cognitive impairment and cardio-vascular symptoms. The pathophysiology underlying long-onset COVID remains poorly understood.

Methods: Blood was collected from patients with Long COVID with symptoms duration > 3 months (LC) (n=30), previously infected by SARS-CoV-2 but without persistent symptoms (resolved COVID-19 (CR), n=10), or healthy donor (n=20). MK frequency in blood was quantified by flow cytometry. Platelets and blood MKs were analysed for microclots, the presence of Spike protein and SARS-CoV-2 RNA by in situ hybridization and immunodetection visualized by confocal microscopy. Spike and serotonin were quantified in plasma.

Results: The frequency of CD41+ MKs in peripheral blood mononucleated cells (PBMCs) was significantly higher than healthy donors (0.28±0.05 versus 0.03±0.02) as a sign of MK infection, as we previously shown in acutely infected individuals with SARS-CoV-2 in platelets. Accordingly, in all samples analyzed, circulating MK in Long COVID sheltered both Spike and SARS-CoV-2 ssRNA, but also dsRNA suggestive of viral replication. These infected MKs produced blood platelets that contain also P Spike and SARS-CoV-2 ssRNA. Platelets microclots were detected in all tested Long COVID patients. Spike protein was detected at the pg level in 30 % of analyzed plasma from Long COVID but not CR individuals. The level of serotonin in platelet and of tryptophan hydroxylase-1 (TPH-1), the enzyme that regulates serotonin synthesis decreased significantly (p<0.0001) in blood of Long COVID patients compared to CR individuals.

Conclusions: In patients developing Long COVID, SARS-CoV-2 persists and replicates in MKs producing virus-containing platelets. The presence of spike in plasma might be an additional sign of viral persistence that could be used as a Long COVID biomarker. The presence of the virus could lead to abnormal platelet activation and the formation of microclots, which would contribute to the various symptoms and to deregulation of serotonin uptake, contributing to the neurocognitive symptoms observed in long-onset COVID.

Source: Feifan He, Boxin Huang, Andrea Cottignies-Calamarte, Wiem Bouchneb, Agathe Goubard, Faroudy Boufassa, Jacques Callebert, Dominique Salmon, Morgane Bomsel. Persistence of SARS-CoV-2 in Platelets and Megakaryocytes in Long COVID. The Conference on Retroviruses and Opportunistic Infections (CROI), March 3-6, 2024 | Denver, Colorado. https://www.croiconference.org/abstract/persistence-of-sars-cov-2-in-platelets-and-megakaryocytes-in-long-covid/ 

Bioimpedance spectroscopy characterization of Myalgic Encephalomyelitis/ Chronic Fatigue Syndrome (ME/CFS) peripheral blood mononuclear cells

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.

Source: Sara Martinez Rodriguez, Alberto Olmo Fernandez, Daniel Martin Fernandez, Isabel Martin-Garrido. Bioimpedance spectroscopy characterization of Myalgic Encephalomyelitis/ Chronic Fatigue Syndrome (ME/CFS) peripheral blood mononuclear cells. Biomedical Letters, Volume 9, Issue 2: 121-128. http://thesciencepublishers.com/biomed_lett/v9i2abstract6.html (Full text available as PDF file)

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.

Source: Alberto Olmo Fernández, Sara Martínez Rodríguez, Daniel Martín Fernández, et al. Bioimpedance spectroscopy characterization of osmotic stress processes in Myalgic Encephalomyelitis / Chronic Fatigue Syndrome (ME-CFS) blood samples. Authorea. July 11, 2023.
DOI: 10.22541/au.168909663.38868952/v1 https://www.authorea.com/doi/full/10.22541/au.168909663.38868952/v1 (Full text)

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)