A multiplex immunoassay gives different results than singleplex immunoassays which may bias epidemiologic associations

Koning, Lawrence de; Liptak, Cary; Shkreta, Aida; Bradwin, Gary; Hu, Frank B.; Pradhan, Aruna D.; Rifai, Nader; Kellogg, Mark D.

doi:10.1016/j.clinbiochem.2012.04.006

Cited by 22 publications

(15 citation statements)

References 10 publications

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“…It has already been stressed out that the comparison of absolute cytokine concentration should be attempted only if used reagents were obtained from the same supplier (Khan et al, 2004), although even singleplex assays compared to multiplex assays from the same company can yield significantly different results (de Koning et al, 2012;Huttunen et al, 2014). Indeed, we confirmed that the absolute values of the proteins detected were different between the two tested technologies.…”

Section: Discussionsupporting

confidence: 69%

How to: Measuring blood cytokines in biological psychiatry using commercially available multiplex immunoassays

Belzeaux

Lefebvre

Lazzari

et al. 2017

Psychoneuroendocrinology

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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. Dear Editor,We thank you and the Editor in chief, Robert Dantzer, for reaching the conclusion to ultimately publish our manuscript. Following your suggestion, we changed the tiltle to: "How to: Measuring blood cytokines in biological psychiatry using commercially available multiplex immunoassays".We were concerned by the persistent negative comments of Reviewer#3, stressing out that our comparison between the two platforms we have worked with suffers a number of theoretical and technical limitations because...

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Section: Discussionsupporting

confidence: 69%

How to: Measuring blood cytokines in biological psychiatry using commercially available multiplex immunoassays

Belzeaux

Lefebvre

Lazzari

et al. 2017

Psychoneuroendocrinology

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“…3) Control of confounders is extremely challenging, since blood cytokine and soluble cytokine receptor levels are affected by a large number of factors such as age, sex, socioeconomic status, acute exercise, excessive caffeine use, BMI, smoking, sleep quality and habits, alcohol use, psychotropic medications, and medical comorbidities (O'Connor et al, 2009b). 4) Different immunoassay techniques may yield different results (de Koning et al, 2012). 5) Genetic and epigenetic effects may strongly influence individual reactivity to inflammatory stimuli (Kim et al, 2013).…”

Section: Discussionmentioning

confidence: 99%

The role of cytokines in the pathophysiology of suicidal behavior

Ganança

Oquendo

Tyrka

et al. 2016

Psychoneuroendocrinology

186

128

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Objective Immune dysregulation has been implicated in depression and other psychiatric disorders. What is less clear is how immune dysregulation can affect risk of suicidal behavior. We reviewed the scientific literature concerning cytokines related to suicidal ideation, suicidal behavior and suicide, and surveyed clinical and neurobiological factors associated with cytokine levels that may modulate effects of inflammation on suicide risk. Methods We searched PubMed, Embase, Scopus and PsycINFO for relevant studies published from 1980 through February, 2015. Papers were included if they were written in English and focused on cytokine measurements in patients with suicidal behaviors. Results The literature search yielded 22 studies concerning cytokines and suicidal ideation, suicide attempts or suicide completion. The most consistent finding was elevated interleukin (IL)-6, found in 8 out of 14 studies, in CSF, blood, and postmortem brain. In one study, IL-6 in CSF was also found to be higher in violent than nonviolent attempters and to correlate with future suicide completion. Low plasma IL-2 was observed in 2 studies of suicide attempters, while divergent results were seen for tumor necrosis factor (TNF)-α, interferon (IFN)-γ, transforming growth factor (TGF)-β, IL-4, and soluble Il-2 receptors. Conclusions Given the complexity suggested by the heterogenous cytokine findings, putative mediators and moderators of inflammation on suicidal behavior merit further study. Elevated IL-6 was the most robust cytokine finding, associated with suicidal ideation and both nonfatal suicide attempts and suicides. Future studies should evaluate the predictive value of high IL-6, consider how this may alter brain function to impact suicidal behavior, and explore the potential beneficial effects of reducing IL-6 on suicide risk.

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“…It is well known that in addition to selected sample material, the cytokine profile and cytokine concentrations can be affected by personal differences such as health status, gender, age and ethnicity, all adding to the variability in the results of cytokine analysis [9], [11], [13], [14] and [19]. Also the differences between commercial assays of separate manufacturers and analysis methods can make the results from different studies incomparable [20], [21] and [22]. Other factors possibly causing variability to the results include lot-to-lot variation, different analysing laboratories, different storage conditions and handling of biological samples [19], [20] and [23].…”

Section: Discussionmentioning

confidence: 99%

The effect of assay type and sample matrix on detected cytokine concentrations in human blood serum and nasal lavage fluid

Huttunen

Tiihonen

Roponen

et al. 2014

Journal of Pharmaceutical and Biomedical Analysis

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Cytokine concentrations in biological fluids are widely used markers for activation of immunological processes. Confirming the reproducibility of measurements is important, especially in longitudinal or multicenter studies where time between analyses or different analyzing laboratories increases the intra-assay variability. In this study, the reproducibility of the cytokine analysis conducted with different assay platforms was studied by comparing the results of two cytokines [interleukin (IL)-6 in serum and nasal lavage fluid (NAL) and IL-8 in NAL] analyzed with Meso Scale Discovery (MSD) ultra-sensitive single and multiplex assay kits (n=76). In addition, the difference in cytokine levels between two biological sample matrices was studied by comparing the results of altogether 9 cytokines [IL-6, IL-2, IL-8, IL12p70, IL-1β, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN)γ, IL-10 and tumor necrosis factor (TNF)α] measured from serum and NAL of the same study subjects (n=460). The results show that the cytokine concentrations analyzed with single and multiplex assays are concordant but not equal. Comparison of the different matrices revealed that cytokine concentrations in serum do not correspond with concentrations detected in nasal lavage fluid. It can be concluded that comparability of the results from single and multiplex analysis of cytokines is high, but the concentrations should not be compared directly with each other. The differences between concentrations analyzed from serum and nasal lavage fluid indicate that the levels are specific for each matrix and represent distinct immunological conditions.

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A multiplex immunoassay gives different results than singleplex immunoassays which may bias epidemiologic associations

Cited by 22 publications

References 10 publications

How to: Measuring blood cytokines in biological psychiatry using commercially available multiplex immunoassays

How to: Measuring blood cytokines in biological psychiatry using commercially available multiplex immunoassays

The role of cytokines in the pathophysiology of suicidal behavior

The effect of assay type and sample matrix on detected cytokine concentrations in human blood serum and nasal lavage fluid

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