Development and validation of a self-prepared phenol red thread test

Abstract

Purpose : The phenol red thread test (PRTT) is the preferred diagnostic tool to quantify aqueous tear production in small animals, particularly rodents, due to the ease of placement and short testing time. Despite its benefits, commercially available PRTTs have been discontinued, creating a need for an alternative source. Therefore, we developed a self-prepared PRTT and tested it both in vitro and in vivo.

Methods : Cotton thread was soaked in phenol red, a pH indicator, dried, cut and sterilized. To test its wicking properties, the thread was inserted into solutions of various pH, flanking the pH of healthy tears, for different time intervals. We also assessed the diagnostic utility of the self-prepared PRTT in vivo by comparing it to the commercial product. PRTTs were performed on mice 2-3 times utilizing the commercial and the self-prepared threads with at least 24 hours between tests to allow normalization of the tear film. The PRTTs were performed in wildtype mice (n=10) and acyl-coA wax alcohol acyltransferase 2 knockout mice (Awat2 KO, n=6), a model of evaporative dry eye disease.

Results : Two batches of self-prepared PRTT were generated and their wicking characteristics were compared. No significant differences in solution absorption were identified at any time point across the pH solutions for batch 1 and only one difference was identified for batch 2 (pH 7.4 vs 7.8 at 5 seconds; mixed effects analysis, P=0.0292). When comparing the first and second batches, they showed very similar wicking kinetics with only two significant differences identified (pH 7.6 at 40 seconds and pH of 7.8 at 35 seconds; mixed effects analysis, P<0.01). In vivo, the self-prepared PRTT yielded similar measurements to the commercial PRTT in wildtype (Bland-Altman; bias: 1.155, 95% limits of agreement: -2.253 to 4.564) and Awat2 KO mice (Bland-Altman; bias: 1.673, 95% limits of agreement: -2.517 to 5.862). Additionally, a significant increase in tear volume was identified in the Awat2 KO mice with both the commercial (t-test, P=0.0151) and self-prepared PRTT (t-test, P=0.0018).

Conclusions : The self-prepared PRTT demonstrated negligible differences in wicking characteristics across solutions and between batches, highlighting the reproducibility of this method. Additionally, the self-prepared PRTT provides statistically similar measurements to the commercial thread indicating that it can serve as a valid alternative for the commercial version.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.