Tear Samples for Protein Extraction: Comparative Analysis of Schirmer's Test Strip and Microcapillary Tube Methods

Abstract

Introduction: Tear sampling is an attractive option for collecting biological samples in ophthalmology clinics, as it offers a non-invasive alternative to other invasive techniques. However, there are many tear sampling methods still in consideration. This study explores the suitability of Schirmer's test strip and microcapillary tube as reliable and satisfactory methods for tear sampling.

Methods: Tear samples were collected from eight healthy volunteers using the standard Schirmer's test strip method with or without anesthesia and microcapillary tubes. The total tear protein concentrations were analyzed via spectrophotometry and bicinchoninic acid (BCA) protein assay. The protein profile was determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal wetting length of Schirmer's strip and suitable buffer solutions were compared. Discomfort levels reported by participants and the ease of execution for ophthalmologists were also evaluated.

Results: Tear samples exhibited typical protein profiles as shown by SDS-PAGE. The mean total protein obtained from an optimum wetting length of 20 mm using Schirmer's strip without anesthesia in phosphate-buffered saline (PBS) yielded substantial quantities of protein as measured by nanophotometer (220.20 ± 67.43 µg) and the BCA protein assay (210.34 ± 59.46 µg). This method collected a significantly higher quantity of protein compared to the microcapillary tube method (p=0.004) which was much more difficult to standardize. The clinician found it harder to utilize microcapillary tubes, while participants experienced higher insecurity and less discomfort with the microcapillary tube method. PBS used during the tear protein extraction process eluted higher tear protein concentration than ammonium bicarbonate, although the difference was not statistically significant. Using anaesthesia did not ease the sampling procedure substantially and protein quantity was maintained.

Conclusion: Good quality and quantity of protein from tear samples were extracted with the optimized procedure. Schirmer's strip test in the absence of local anesthesia provided a standard, convenient, and non-invasive method for tear collection.

Keywords: microcapillary tube; protein quantitation; schirmer’s test strip; sodium dodecyl sulphate-polyacrylamide gel electrophoresis (sds-page); tear sampling.

Figure 1. SDS-PAGE analysis of tear proteins collected using Schirmer's test strip and microcapillary tube.

Lane L: BLUltra pre-stained standard protein marker (6.5-270 kDa) (GeneDireX, Taiwan); S: Tears collected using Schirmer's test strip without anesthesia in PBS (N=8); S’: Tears collected using Schirmer's test strip with anesthesia in PBS (N=5); S*: Tear collection using Schirmer's test strip without anesthesia in NH₄HCO₃ (N=2); M: Tears collected using microcapillary tube (N=7) Lane 1: 10 mm strip; Lane 4: 30 mm strip; Lane 2, 3, 5, 9-20: 20 mm strip; Lane 6-8, Lane 21-24: Microcapillary collected tear (N=7). PBS: phosphate-buffered saline; SDS-PAGE: sodium dodecyl sulphate-polyacrylamide gel electrophoresis

Figure 2. The dispersion of the standard curve and its equation for average absorbance obtained at 570 nm using Pierce BCA Protein Assay Kit for BSA.

BSA: bovine serum albumin Manufacturer details: Pierce BCA Protein Assay Kit, Thermo Fisher Scientific, Waltham, Massachusetts, United States