A comparison of biofluid cytokine markers across platform technologies: Correspondence or divergence?

Abstract

Background: Quantification of biofluid cytokines is a rapidly growing area of translational research. However, comparability across the expanding number of available assay platforms for detection of the same proteins remains to be determined. We aimed to directly compare a panel of commonly measured cytokines in plasma of typically aging adults across two high sensitivity quantification platforms, Meso Scale Discovery high performance electrochemiluminiscence (HPE) and single-molecule immunosorbent assays (Simoa) by Quanterix.

Methods: 57 community-dwelling older adults completed a blood draw, neuropsychological assessment, and brain MRI as part of a healthy brain aging study. Plasma samples from the same draw dates were analyzed for IL-10, IP-10, IL-6, TNFα, and IL-1β on HPE and Simoa, separately. Reliable detectability (coefficient of variance (CV) < 20% and outliers 3 interquartiles above the median removed), intra-assay precision, absolute concentrations, reproducibility across platforms, and concurrent associations with external variables of interest (e.g., demographics, peripheral markers of vascular health, and brain health) were examined.

Results: The proportion of cytokines reliably measured on HPE (87.7-93.0%) and Simoa (75.4-93.0%) did not differ (ps > 0.32), with the exception of IL-1β which was only reliably measured using Simoa (68.4%). On average, CVs were acceptable at <8% across both platforms. Absolute measured concentrations were higher using Simoa for IL-10, IL-6, and TNFα (ps < 0.05). HPE and Simoa shared only small-to-moderate proportions of variance with one another on the same cytokine proteins (range: r = 0.26 for IL-10 to r = 0.64 for IL-6), though platform agreement did not dependent on cytokine concentrations. Cytokine ratios within each platform demonstrated similar relative patterns of up- and down-regulation across HPE and Simoa, though still significantly differed (ps < 0.001). Supporting concurrent validity, all 95% confidence intervals of the correlations between cytokines and external variables overlapped between the two platforms. Moreover, most associations were in expected directions and consistently so across platforms (e.g., IL-6 and TNFα), though with several notable exceptions for IP-10 and IL-10.

Conclusions: HPE and Simoa showed comparable detectability and intra-assay precision measuring a panel of commonly examined cytokine proteins, with the exception of IL-1β which was not reliably detected on HPE. However, Simoa demonstrated overall higher concentrations and the two platforms did not show agreement when directly compared against one another. Relative cytokine ratios and associations demonstrated similar patterns across platforms. Absolute cytokine concentrations may not be directly comparable across platforms, may be analyte dependent, and interpretation may be best limited to discussion of relative associations.

Keywords: Immune activation; Meso Scale Discovery; Plasma markers; Quanterix; Typical aging.

Figure 1

Proportion of samples reliably measured across cytokine markers on high performance electrochemiluminescence (HPE) and single molecule array (SIMOA) platforms did not differ, with the exception of IL1β.

Figure 2

Intra-assay precision of plasma cytokine measurements by high performance electrochemiluminescence (HPE) and single molecule array (SIMOA) platforms. Note. CV = coefficient of variance; *p<0.05.

Figure 3

Absolute concentrations (pg/mL) differ between high performance electrochemiluminescence (HPE) and single molecule array (SIMOA) platform measurements for IL-10, IL-6, and TNFα Note. *p<0.05.

Figure 4

Cytokine concentrations are significantly, but not strongly correlated with each other and demonstrate significant differences as quantified by high performance electrochemiluminiscence (HPE) versus single molecule array (Simoa) platforms. Note. A) Scatterplots depicting agreement between HPE (x-axis) and SIMOA (y-axis); B) Bland-Altman plots illustrating platform differences. The y-axis represents the mean differences between HPE and SIMOA, while the x-axis depicts the average between the platforms. The solid horizontal lines indicate the average difference and the dashed horizontal lines represent the 95% confidence intervals.

Figure 5

Relative ratios of cytokine concentrations demonstrate a similar pattern of increases and decreases, but still significantly differ across platforms. Note. Y-axis represents cytokine concentration ratios (log transformed + 3 in pg/mL). HPE = high performance electrochemiluminiscence; Simoa = Quanterix single molecule array. All ratios values are significantly different between MSD HPE and Quanterix Simoa (ps<0.001).

Figure 6

Correlations (r and 95% error bars) between each measured cytokine and external variables of interest across high performance electrochemiluminiscence (HPE) and single molecule array (SIMOA) platforms. Note. MMSE = Mini Mental State Examination; Corpus Call FA = corpus callosum fractional anisotropy; CRP = C-reactive protein; HOMA-IR = homeostatic model assessment of insulin resistance; BMI = body mass index.