Immuno oncology panel

An array of growth factors, cytokines, and chemokines was selected for analysis based on previous evidence suggesting their involvement in CRS, olfactory dysfunction, or inflammation/remodeling. To comprehensively evaluate these indicators as possible, we chose to measure protein levels using the Olink Immuno-oncology panel. This panel employs the Proximity Extension Assay (PEA) technology from Olink Proteomics AB (Uppsala, Sweden). The protein levels were determined following the manufacturer’s instructions and are presented in normalized protein expression units as NPX values [20 (link)]. Subsequent differential protein analysis was based on an NPX matrix representing relative quantitative protein levels. Some values including high proportions (>33%) below the lower limit of detection were excluded from these analyses (see Table 2).
Furthermore, selected markers were quantified using a user-mixed magnetic Luminex assay kit obtained from R&D Systems (Minneapolis, MN, USA). The total protein concentration in the samples was determined using the Bradford assay (Thermo Fisher Scientific).

The serum proteome was characterized across 4 sequential time points (days 0, 4, 14, 28) in 52 patients using a 92-protein multiplex proximity extension assay from the Olink platform (“Immuno Oncology Panel,” Olink Bioscience). Experiments were performed as previously described.^{44 (link),45 (link)} Briefly, olignonucelotide-labeled monoclonal or polyclonal antibodies (PEA probes) were used to bind target proteins in a pairwise manner, thereby preventing all cross-reactive events. Upon binding, the oligonucleotides come in close proximity and hybridize, followed by extension, generating a unique sequence used for the digital identification of the specific protein assay.
A linear mixed model (LMM) was applied per protein that tested differences of serum proteins by patient group (infection versus CRS^only control) and time point. To account for potential confounding, age and sex were included as fixed effects, while a random effect per patient was included to account for potentially differing baselines. Regression models were fitted using the R-packages lme4 and lmerTest, while plots were generated using the OlinkAnalyze and ggplot2 packages. Protein levels were expressed in Normalized Protein eXpression (NPX) units, which were derived from Ct values. Because NPX is expressed in a log2 scale, a 1 NPX difference translates into a doubling of protein concentration.