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Multiplex Cytokine Profiling of Stimulated Mouse Splenocytes Using a Cytometric Bead-based Immunoassay Platform
Published on: November 9, 2017
How to: Measuring blood cytokines in biological psychiatry using commercially available multiplex immunoassays
Raoul Belzeaux1, Marie-Noëlle Lefebvre2, Anne Lazzari3
1Aix-Marseille Univ., CNRS, CRN2M-UMR7286, Marseille, France; Pôle de Psychiatrie, Assistance Publique-Hôpitaux de Marseille, Marseille, France; Fondation FondaMental, Fondation de Recherche et de Soins en Santé Mentale, Créteil, France.
Insights
Cytokine stability in blood is crucial for psychiatric biomarker discovery. The V-PLEX assay showed better reproducibility and consistency than Luminex for detecting cytokines in major depressive episodes.
Area of Science:
- Neuroscience
- Immunology
- Biomarker Discovery
Background:
- Cytokines play a role in psychiatric disorders, prompting research into their use as biomarkers.
- Previous studies have not sufficiently confirmed the stability of blood cytokine levels in healthy individuals over time.
- Multiplex assays offer advantages for biomarker identification but require validation for sensitivity and reproducibility in neuropsychiatric patients.
Purpose of the Study:
- To compare the performance of two multiplex immunoassay platforms (Luminex and V-PLEX) for quantifying 31 cytokines, chemokines, and growth factors.
- To assess the stability and reproducibility of these cytokine measurements in individuals with major depressive episodes (MDE) and healthy controls over 30 weeks.
- To evaluate the correlation between serum and plasma measurements and compare with a gold standard ELISA assay.
Main Methods:
- Comparison of Luminex (bead-based) and V-PLEX (electro-chemiluminescence-based) multiplex assays.
- Quantification of 31 analytes in serum and plasma from MDE patients and healthy controls over 30 weeks.
- Assessment of linearity, reproducibility, correlation between sample types, and comparison with ELISA for IL-6.
Main Results:
- Both platforms showed low coefficients of variability in calibration curves, with V-PLEX demonstrating better linearity.
- V-PLEX generally offered better intra-assay reproducibility for the 16 analytes detected by both assays.
- Consistent results between serum and plasma were more frequent with V-PLEX, which also showed better agreement with ELISA for IL-6.
Conclusions:
- The V-PLEX platform appears more suitable for cytokine profiling in psychiatric research due to better reproducibility and consistency.
- Significant intra-individual variability was observed for certain cytokines (e.g., IFN-γ, IL-6), highlighting challenges in biomarker stability.
- Caution is advised when translating multiplex cytokine profiling results into psychiatric biomarker discovery due to assay performance and analyte variability.
Abstract:
Cytokines produced by both immune and non-immune cells are likely to play roles in the development and/or progression of psychiatric disorders. Indeed, many investigators have compared the blood cytokine levels in psychiatric patients with those of healthy controls or monitored their levels in patients during disease progression to identify biomarkers. Nevertheless, very few studies have confirmed that such cytokines remain stable in healthy individuals through periods of weeks and months. This is an important issue to consider before using blood cytokine levels as biomarkers of disease traits, disease state, or treatment response. Although multiplex assay technology represents an advance in identifying biomarkers because it allows simultaneous examination of large panels of analytes from a small volume of sample, it is necessary to verify whether these assays yield enough sensitivity and reproducibility when applied to the blood from neuropsychiatric patients. Therefore, we compared two multiplex immunoassays, the bead-based Luminex® (Bio-Rad) and the electro-chemiluminescence-based V-plex® (MesoScaleDiscovery), for the detection and quantification of 31 cytokines, chemokines and growth factors in both the sera and plasma of patients with major depressive episodes (MDE) and age- and sex-matched healthy control subjects during a 30-week period. Although both platforms exhibited low coefficients of variability (CV) between the duplicates in the calibration curves, the linearity was better in general for the V-PLEX® platform. However, neither platform was able to detect the absolute values for all of the tested analytes. Among the 16 analytes that were detected by both assays, the intra-assay reproducibility was in general better with the V-PLEX® platform. Although it is not a general rule that the results from sera and plasma will be correlated, consistent results were more frequent with the V-PLEX® platform. Furthermore, the V-PLEX® results were more consistent with the gold standard ELISA simplex assay for IL-6 in both sera and plasma. The intra-individual variability of the measurements, among the sera and plasma for the 4 samples harvested from each healthy individual, was low for Eotaxin, G-CSF, IL-4, IL-7, IL-9, IL-12p40, IL-12p70, IL-15, MIP-1β, PDGF-BB, TNF, TNF-β and VEGF, but intermediate or high for IFN-γ, IL-6, IL-8, IL-10, and IP10. Together, these data suggest that extreme caution is needed in translating the results of multiplex cytokine profiling into biomarker discovery in psychiatry.
