AH specimens were collected by anterior chamber paracentesis with the use of a 30-gauge needle on a 1 mL tuberculin syringe prior to initiation of the concomitant procedure. Specimens were stored in a freezer at 80° C until they were sent to Olink Proteomics (Uppsala, Sweden) for analysis. Potential protein biomarkers from the Olink Immuno-Oncology panel (v.3111) were evaluated using a Proximity Extension Assay (PEA) technique, which has been described in detail elsewhere.
12 This particular panel was chosen because of its unique inclusion of a variety of both inflammatory cytokines and growth factors. A full list of potential protein biomarkers included in this panel (92 in total) and associated assay validation data (e.g., limit of detection [LOD], lower and upper limits of quantification, within- and between-run precision coefficient of variation) can be found on the manufacturer's website (
https://www.olink.com). The final protein concentration output from these assays is reported in Normalized Protein eXpression (NPX) values. The NPX is an arbitrary unit on a log base 2 scale wherein higher NPX values correlate with higher protein concentrations. For example, a 1-point difference in an NPX value is equivalent to a twofold change in protein concentration.
Protein expression data results from 60 AH specimens from 60 patients were obtained. One specimen from a patient with DM with PDR failed the manufacturer's quality control test and was excluded. Thirty-two potential protein biomarkers were found to have a high frequency (50% or greater) of NPX values below the LOD and were not considered for data analysis in accordance with the manufacturer's recommendations. These included the following: monocyte chemotactic protein 3, CD40 ligand, interleukin-1 alpha, natural killer cell receptor, pro-epidermal growth factor, adhesion G-protein–coupled receptor G1, cytotoxic and regulatory T-cell molecule, fibroblast growth factor 2, mucin-16, endothelial nitric oxide synthase, interleukin-2, granzyme H, killer cell immunoglobulin-like receptor 3DL1, tumor necrosis factor ligand superfamily member 14, programmed cell death protein 1, fas antigen ligand, T-cell-specific surface glycoprotein, interleukin-5, CD70 antigen, interleukin-10, arginase-1, natural cytotoxicity triggering receptor 1, stromal cell-derived factor 1, interferon gamma, lysosome-associated membrane glycoprotein 3, programmed cell death 1 ligand 2, interleukin-4, lymphocyte activation gene 3 protein, interleukin-12 receptor subunit beta-1, interleukin-13, tumor necrosis factor, and natural killer cells antigen CD94. As a result, the final data set described 60 potential protein biomarkers in 59 aqueous humor specimens from 59 patients. All remaining potential protein biomarker concentrations with NPX values below the LOD were included in the final analysis; they were neither replaced with a specific value nor excluded to increase statistical power and reduce distribution skew.