Pre-analytical sample handling effects on tear fluid protein levels.

Tear fluid is emerging as a source of non-invasive biomarkers, both for ocular and systemic conditions. Accurate quantification of tear proteins can be improved by standardizing methods to collect and process tear fluid. The aim of this study was to determine sample handling factors that may influence the tear protein biomarker profile. Tear fluid was collected using Schirmer's strips. Tear proteins were extracted by elution through centrifugation. Total protein content was determined using the bicinchoninic acid assay. Key concepts that apply to the entire sample processing cycle are tear sampling, tear storage, protein extraction and data normalization. Differences in wetting or migration length were observed between Schirmer's strips from different manufacturers, and between protein-free and protein-rich solutions. One unit of migration length (mm) did not correspond to one unit of volume (µL). A positive correlation (r = 0.6671, p < 0.0001) was observed between migration length and total tear protein content. The most beneficial storage conditions were strips that were not stored (+ 21.8%), or underwent 'wet' storage (+ 11.1%). Protein recovery was the highest in 400 µL extraction buffer and independent of protein molecular weight. This study helps to explain inter- and intra-variability that is often seen with tear biomarker research. This information is critical to ensure accuracy of test results, as tear biomarkers will be used for patient management and in clinical trials in the near future. This study also highlights the need for standardization of Schirmer's strip manufacturing, tear fluid processing and analyte concentration normalization.

Evaluation of pre-processing methods for tear fluid proteomics using proximity extension assays.

Tear fluid forms a potential source for biomarker identification, and can be minimal invasively collected via Schirmer strips. The lack of knowledge on the processing of Schirmer strips however complicates the analysis and between-study comparisons. We studied two different pre-processing methods, specifically the use of punches of the strip versus elution of the strip in a buffer. Tear fluid filled Schirmer strips were collected from 5 healthy participants, and divided into two halves over the length of the strip. In either part, punches or eluates were obtained from 4 different locations, from the first part touching the eye (head) to the end, to assess the protein distribution along the strips. The levels of 92 inflammatory proteins were measured in the punches/eluates using proximity extension assays. The punch method yielded higher protein detectability compared to the elution method (76% vs 66%; p ≤ 0.001). Protein expression level was found to be slightly higher in the head of the strip, however, 3 out of 5 punches from the head failed quality control. Protein expression levels over the remaining parts of the strips were similar. Our study showed beneficial use of punches of any part of the strip except the head in future biomarker research.