Performance of Multiplex Commercial Kits to Quantify Cytokine and Chemokine Responses in Culture Supernatants from <em>Plasmodium falciparum</em> Stimulations

<div><h3>Background</h3><p>Cytokines and chemokines are relevant biomarkers of pathology and immunity to infectious diseases such as malaria. Several commercially available kits based on quantitative suspension array technologies allow the profiling of multiple cytokines and chemokines in small volumes of sample. However, kits are being continuously improved and information on their performance is lacking.</p> <h3>Methodology/Principal Findings</h3><p>Different cytokine/chemokine kits, two flow cytometry-based (eBioscience® FlowCytomix™ and BD™ Cytometric Bead Array Human Enhanced Sensitivity) and four Luminex®-based (Invitrogen™ Human Cytokine 25-Plex Panel, Invitrogen™ Human Cytokine Magnetic 30-Plex Panel, Bio-Rad® Bio-Plex Pro™ Human Cytokine Plex Assay and Millipore™ MILLIPLEX® MAP Plex Kit) were compared. Samples tested were supernatants of peripheral blood mononuclear cells of malaria-exposed children stimulated with <em>Plasmodium falciparum</em> parasite lysates. Number of responses in range that could be detected was determined and reproducibility of duplicates was evaluated by the Bland-Altman test. Luminex® kits performed better than flow cytometry kits in number of responses in range and reproducibility. Luminex® kits were more reproducible when magnetic beads were used. However, within each methodology overall performance depended on the analyte tested in each kit. Within the Luminex® kits, the Invitrogen™ with polystyrene beads had the poorer performance, whereas Invitrogen™ with magnetic beads had the higher percentage of cytokines/chemokines with both readings in range (40%), followed by Bio-Rad® with magnetic beads (35%). Regarding reproducibility, the Millipore™ kit had the highest percentage (60%) of cytokines/chemokines with acceptable limits of agreement (<30%), followed by the Invitrogen™ with magnetic beads (40%) that had tighter limits of agreement.</p> <h3>Conclusions/Significance</h3><p>Currently available kits for cytokine and chemokine quantification differ in reproducibility and concentration range of accurate detection. Luminex®-based kits with magnetic beads perform the best. Data highlights the importance of testing different kits before each study to choose the most appropriate, depending on the priority of the cytokines assessed.</p> </div

Proportion of duplicate readings in range.

<p>. (not tested).</p><p>Proportions of both readings in range higher than 75% are highlighted in bold.</p><p>INV-MAG: Invitrogen™ kit with magnetic beads.</p

Limits of agreement for each cytokine/chemokine tested.

<p>When differences are <30% the limits of agreement are considered acceptable and are highlighted in bold.</p><p>. (Cytokine/chemokine not tested).</p><p>– (less than 8.1% of both readings in range).</p>*<p>Constant variance p value <0.05.</p>†<p>Constant ratio p value <0.05.</p>‡<p>Ratio is 1 p value <0.05.</p><p>INV-MAG: Invitrogen™ kit with magnetic beads.</p

Mean difference dot plots of IL-10 and IL-2 cytokines for each kit tested.

<p>A) IL-10 and B) IL-2 disagreement plots show the difference between the duplicates against the geometric mean of both values of a sample tested with eBioscience® FlowCytomix™ (FlowCytomix), Millipore™ MILLIPLEX® MAP Plex Kit (Millipore), Bio-Rad® Bio-Plex Pro™ Human Cytokine Plex Assay (Bio-Rad), Invitrogen™ Human Cytokine Magnetic 30-Plex Panel (INV-MAG) and BD™ Cytometric Bead Array Human Enhanced Sensitivity kit (BD CBA). The middle line is the mean difference and the two extreme lines are the limits of agreement (values are truncated to the first decimal) calculated by Bland-Altman test.</p

Correlations of serum biomarker concentrations and IgM and IgG levels against <i>B</i>. <i>bacilliformis</i>.

<p>Only biomarkers with statistical significant associations with antibody levels are shown. P-values were computed through unadjusted linear regressions. The grey area shows the 95% confidence interval for predictions from the linear model.</p

Epidemiologic and demographic characteristics of study participants stratified by RT-PCR, IgM and IgG results.

<p>Epidemiologic and demographic characteristics of study participants stratified by RT-PCR, IgM and IgG results.</p

Serum biomarker concentrations according to <i>B</i>. <i>bacilliformis</i> IgM and IgG serostatus.

<p>Boxplots illustrate the 25^th and 75^th quartiles, and whiskers the percentile 10–90 of biomarkers significantly associated with IgM or IgG seropositivity. P-values were computed through unadjusted linear regressions.</p

Correlations of eotaxin and EGF concentrations with <i>B</i>. <i>bacilliformis</i> bacteremia by RT-PCR.

<p>Scatter plots of subjects with detectable bacteremia by RT-PCR. Only biomarkers with statistical significant correlations with antibody levels are shown. <i>r</i>_s and P-values were computed through Spearman rank correlations. The grey area shows the 95% confidence interval for predictions from the linear model.</p

Spearman correlations between biomarker concentrations and IgM and IgG levels for biomarkers found to be associated with antibody responses.

<p>Spearman correlations between biomarker concentrations and IgM and IgG levels for biomarkers found to be associated with antibody responses.</p

Combinations of markers associated with detectable <i>B</i>. <i>bacilliformis</i> bacteremia obtained through partial least squares discriminant analysis (PLS-DA).

<p>(A) Graphs of marker loadings to the 3 components of the PLS-DA. Bars quantify the importance (loadings) of each marker for the specific PLS-DA components that were significantly associated with RT-PCR results. Biomarkers that substantially contributed to the components (loadings > |0.3|) are highlighted in black. (B) PLS-DA plots representing each sample (dots) with respect the 3 first PLS-DA components.</p