Validation of the Flush Method as an Alternative to Basal or Reflex Tear Collection

Materials and Methods: Total protein content (TPC) and immunoglobulin A (IgA) concentrations were determined in the basal, reflex, and flush tears of 16 healthy non-contact lens wearers. The overall protein profile was established using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS). Results: Collection-rates were 4.6 +/-+/- 6.7 mu mu l/min, 13.9 +/-+/- 11.1 mu mu l/min, and 25.7 +/-+/- 12.4 mu mu l/min for the basal, reflex, and flush tears, respectively. TPC was 7.14 +/-+/- 2.22 mg/mL, 6.01 +/-+/- 2.11 mg/mL, and 3.79 +/-+/- 1.51mg/ mL for basal, reflex, and flush tears, respectively, with flush tears being significantly less concentrated than basal (p == 0.001) and reflex (p == 0.008). IgA concentration was 1.04 +/-+/- 0.29 mg/ mL, 0.64 +/-+/- 0.26 mg/mL, and 0.65 +/-+/- 0.23 mg/mL for basal, reflex, and flush tears, respectively, with basal tears being significantly more concentrated (p < 0.001). As a percentage of TPC, IgA represented 19.8 +/-+/- 14.9%, 11.4 +/-+/- 3.9%, and 19.8 +/-+/- 8.7% for basal, reflex, and flush, respectively. The flush was not significantly different to basal (p == 1.00) but significantly greater than reflex (p == 0.02). SDS-PAGE showed the same tear profiles for basal and flush tears. MS identified the most abundant proteins in all tear types. Conclusions: The flush method allows much faster collection than basal secretion sampling but returns essentially the same spectrum of proteins in similar proportions. This behavior is confirmation that the flush technique has utility as a more convenient alternative to basal tear sampling in studies involving composition analysis.