Multisite comparison of high-sensitivity multiplex cytokine assays

Abstract

The concentrations of cytokines in human serum and plasma can provide valuable information about in vivo immune status, but low concentrations often require high-sensitivity assays to permit detection. The recent development of multiplex assays, which can measure multiple cytokines in one small sample, holds great promise, especially for studies in which limited volumes of stored serum or plasma are available. Four high-sensitivity cytokine multiplex assays on a Luminex (Bio-Rad, BioSource, Linco) or electrochemiluminescence (Meso Scale Discovery) platform were evaluated for their ability to detect circulating concentrations of 13 cytokines, as well as for laboratory and lot variability. Assays were performed in six different laboratories utilizing archived serum from HIV-uninfected and -infected subjects from the Multicenter AIDS Cohort Study (MACS) and the Women's Interagency HIV Study (WIHS) and commercial plasma samples spanning initial HIV viremia. In a majority of serum samples, interleukin-6 (IL-6), IL-8, IL-10, and tumor necrosis factor alpha were detectable with at least three kits, while IL-1β was clearly detected with only one kit. No single multiplex panel detected all cytokines, and there were highly significant differences (P < 0.001) between laboratories and/or lots with all kits. Nevertheless, the kits generally detected similar patterns of cytokine perturbation during primary HIV viremia. This multisite comparison suggests that current multiplex assays vary in their ability to measure serum and/or plasma concentrations of cytokines and may not be sufficiently reproducible for repeated determinations over a long-term study or in multiple laboratories but may be useful for longitudinal studies in which relative, rather than absolute, changes in cytokines are important.

Fig. 1.

Detection frequency of circulating serum levels of cytokines by lab and lot in multiplex assays. Percentage of serum samples (n = 36) with detectable levels of the indicated cytokine in the relaxed data set. White and black bars, assay data from first and second lots, respectively, for each manufacturer's kit within each laboratory; NI, cytokines not included on each kit.

Fig. 2.

Median and range of serum concentrations of selected cytokines by lab and lot in multiplex assays. Box plots showing median (vertical lines in the boxes) and 25th to 75th percentiles of serum concentrations of IL-10 (A), TNF-α (B), and IL-6 (C) from different lots and different labs in 36 subjects. The absence of a median line within a box indicates that at least 50% of samples had <0.1 pg/ml. Whiskers show 1.5 times the interquartile range or the upper limit, whichever was lower; values above 1.5 times the interquartile range are shown as individual data points. White and black bars, first and second lots, respectively, for each manufacturer's kit within each laboratory.

Fig. 3.

HIV viral load in longitudinal plasma samples over the course of initial HIV viremia. Plasma HIV load data from three individual commercial plasma donors (circles, squares, and triangles) with samples available before, on (d0, vertical dashed line), and after the first day that an HIV viral load was detectable (>100 copies/ml, horizontal dotted line).

Fig. 4.

Cytokines with consistent increases detected by multiplex assays over the course of initial HIV viremia. Pooled plasma cytokine data from all three donors across all time points are shown as a smoothed plot (solid lines) for IL-8 (A), IL-10 (B), and IFN-γ (C); dashed lines, 95% confidence interval. IL-8 was not included (NI) in the Bio-Rad multiplex panel. Each point indicates the mean plasma cytokine concentration from two lots in the same laboratory for the indicated kit and cytokine, with each of the six laboratories represented by a different symbol (lab A, red diamonds; lab B, blue squares; lab C, green triangles; lab D, orange circles; lab E, purple × signs; lab F, pink inverted triangles). For graphical purposes, samples with undetectable values were plotted as one-half the minimum value used in left censoring or 0.1 pg/ml, whichever was greater.

Fig. 5.

Cytokines with mixed patterns detected by multiplex assays over the course of initial HIV viremia. Pooled plasma cytokine data from all three donors across all time points (as described for Fig. 4) for IL-1β (A), TNF-α (B), and IL-6 (C). Symbols are described in the Fig. 4 legend.