Post-collection processing of Schirmer strip-collected human tear fluid impacts protein content

Abstract

We examine the impact of post-collection sample handling on the protein composition of human tear samples. In particular, we characterize diffusion-based protein extraction from Schirmer strips. These strips of filter paper membrane are the de facto standard for tear fluid collection and storage, with diffusion-based protein elution off the strip being the most widely reported protein extraction strategy. Nevertheless, the diffusion-based protein elution strategy remains uncharacterized regarding downstream functional protein assays. Here, the time-dependence, concentration-dependence, and repeatability of the diffusion-based protein recovery protocol are characterized. Levels of protein irrecoverable from the Schirmer strip and lost during sample handling are isolated and compared for several major tear proteins. Further, the impact of the Schirmer strip and sample handling on the downstream concentration of proteins ranging in molecular weight, surface charge, and surface hydropathicity is quantified. Diffusion-based protein extraction from Schirmer strips was observed to be protein-dependent. Schirmer strips retained tear proteins to varying extents: 14.2% of lysozyme, 9.5% of human serum albumin, 27.7% of secretory IgA, and 30.9% of mucin 4. Tear protein loss during sample handling ranged from 2% (lysozyme) to 41.2% (mucin 4). Strip retention of protein was observed to be associated with protein molecular weight and hydrophobic surface area. Greater sample handling loss was associated with increased hydrophobic surface area of model proteins. Surface charge or surface hydrophilicity was not significantly associated with protein loss. We therefore conclude that, although diffusion-based processing of Schirmer strip-collected tear samples is widely used, these protocols may result in total post-collection protein loss which is considerable, consistent, and protein-dependent. This loss alters the relative and absolute protein concentrations in the sample. A priori prediction of strip-losses for individual proteins does not appear to be facile, based on cursory knowledge of protein surface properties. Thus, we emphasize "spike and recover" control experiments to determine expected elution profiles for target proteins when using diffusion-based protein sample preparation for Schirmer strip-collected tear fluid.

Single unit filter-aided method for fast proteomic analysis of tear fluid

“…It has been shown that 3-h extraction is sufficient to achieve time-independent protein extraction from Schirmer strips [148,149,154]. In our study, we experienced a slight increase in the number of identified proteins with overnight extraction compared with a 4-h extraction.…”

An eye on the dog as the scientist's best friend for translational research in ophthalmology: Focus on the ocular surface

“…41 cellulose filter paper and possess specific characteristics to promote tolerance (5-mm width × 35-mm length, 0.22-mm thick, 20-25 µm porosity, foldable extremity for ease of insertion). 249,257 In addition to their conventional use for aqueous tear assessment, Schirmer strips can retrieve tear fluid for bioanalytical purposes in humans, 182,216,219,220,228,231,258 dogs, 190,209,211,214,215,240,259 and small laboratory species. 244,260,261 For instance, Schirmer strips were used for indepth characterization of proteomics in human 173 and canine tears, 190 and can also successfully recover specific analytes such as cytokines, 200,258 clusterin, 182 and xenobiotics.…”

“…250 On this note, the main limitation of indirect sampling is the "invasiveness" of the technique, at risk of promoting reflex tearing and altering the composition of the tear fluid; indeed, several studies showed variable tear composition between directly and indirectly collected samples, with notable differences in the qualitative and quantitative profiles obtained for tear lipids 221 and tear proteins. 216,220,226,269 Another important drawback is related to the adsorptive properties of Schirmer strips or absorbent sponges, that is, incomplete release of tear compounds following extraction 209,214,257,258,265 ; however, the authors believe this limitation can be minimized/controlled with adequate precautions (see Section 3.3).…”

Development of a highly sensitive and reliable enzyme-linked immunosorbent assay for MUC5AC in human tears extracted from Schirmer strips

“…The Schirmer test is widely used to measure tear volume in multicenter clinical trials because it is easy to apply and does not require special equipment 14,15. This test can also be used to collect tear samples16 for analysis of cytokines,17 nucleic acids,18 and immunoglobulin 19…”