Effects of serum and plasma matrices on multiplex immunoassays

Abstract

Multiplexed fluorescence or electrochemiluminescence immunoassays of soluble cytokines are commonly performed in the context of human serum or plasma, to look for disease biomarkers and to monitor the immune system in a simple and minimally invasive way. These assays provide challenges due to the complexities of the matrix (serum or plasma) and the presence of many cytokines near the limit of detection of the assay. Here, we compare the readout of matched serum and plasma samples, which are generally correlated. However, a subset of cytokines usually have higher levels in serum, and the non-specific background is significantly increased in serum versus plasma. Presumably as a result of this non-specific background, disease-related decreases in low-abundance cytokines can sometimes be detected in plasma but not in serum. We further show, through spike recovery experiments, that both serum and plasma inhibit the readout of many cytokines, with some variability between donors, but with serum causing greater inhibition than plasma in many cases. Standard diluents from different vendors can partially reverse this inhibition to varying degrees. Dilution of samples can also partly overcome the inhibitory effect of the matrix. We also show that dilution is nonlinear and differentially affects various cytokines. Together, these data argue that (1) plasma is a more sensitive matrix for detecting changes in certain low-abundance cytokines; (2) calculation of concentrations in serum or plasma matrices is inherently inaccurate; and (3) dilution of samples should not be assumed to be linear, i.e., all comparisons need to be made among similarly diluted samples.

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

“…Therefore, metaanalyses should distinguish studies of serum samples from the ones that used plasma samples. Moreover, some investigators have reported that the plasma cytokine concentrations were generally higher than those measured in serum due to a higher nonspecific background observed in serum (Chaturvedi et al, 2011;Rosenberg-Hasson et al, 2014;Wong et al, 2008). In our study, we have confirmed such a trend only for the Luminex® platform (Supplementary Table 5), which may explain why the detectability was better for that platform in plasma compared to sera (Tables 2 and 3).…”

“…This could explain, at least in part, the increased sensitivity of the latter. Nevertheless, it has been shown that (i) both serum and plasma inhibit the readouts, (ii) the inhibitory effect of the matrix is partly reversed by dilution, and (iii) this type of inhibition is much higher with the Luminex® system compared to the MSD assays (Rosenberg-Hasson et al, 2014). In addition, as previously noted in a few studies (Dossus et al, 2009;Dupuy et al, 2013;Toedter et al, 2008), the nature of the capture/detection antibody reactivity is crucial.…”

“…Very few studies have compared the serum and plasma concentrations of cytokines from the same individuals (Altara et al, 2015;Chaturvedi et al, 2011;Clendenen et al, 2010;Dossus et al, 2009;Rosenberg-Hasson et al, 2014;Wong et al, 2008). In general, it has been found that these marker measurements are not interchangeable between serum and plasma.…”

“…Among the various multiplex platforms, the fluorescent bead-based immunoassay Luminex® (Bio-Rad) and the electro-chemiluminescence immunoassay V-plex® (Meso Scale Discovery) technologies dominate the market (Rosenberg-Hasson et al, 2014). Thus far, very few investigators have compared these technologies and evaluated the limits of quantification and intra-assay reproducibility for assay of human blood (Breen et al, 2011;Chowdhury et al, 2009;Fu et al, 2010;Nechansky et al, 2008;Toedter et al, 2008).…”

Systemic serum amyloid A as a biomarker for exposure to zinc and/or copper-containing metal fumes

“…The matrix inhibitory effects of serum and plasma matrices on the detection of soluble cytokines using immunoassays has been shown to exist to a much lesser extent in the electro-chemiluminescence platform from MSD, when compared with either magnetic or polystyrene bead Luminex assays. 20 The average spike recovery for SAA in EDTA plasma was 101%, with a coefficient of variation (CV) of 9.3%. The corresponding recovery range was between 75% and 125%.…”