| Purpose. This study compares vendor reagents used for cytometric bead-based assay (CBA) of cytokines in human tears and validates a protocol for use in evaluating punctal occlusion, a treatment for keratoconjunctivitis sicca. It has been proposed that punctal occlusion may alter tear cytokine concentrations based on the facts that: (1) tear clearance, a measure of the balance between tear production and elimination, declines following occlusion and (2) reduced tear clearance and increased levels of pro-inflammatory tear cytokines are correlated in dry eye patients. This study compares tear clearance values and the level of 24 tear cytokines measured by CBA following temporary unilateral punctal occlusion. Methods. Multiple reagent kits from three vendors were compared in parallel. Vendor protocols were modified to accommodate sample volumes and performance measures of precision, recovery, linearity, sample stability, and tear collection effects were evaluated. Twenty-three subjects received superior and inferior unilateral intracanalicular punctal occlusion with six-month dissolvable plugs. Nonstimulated tear samples were collected from both eyes by micropipette (stored at -80 C) and tear clearance was measured using the Standardized Visual Scale Test prior to occlusion and again at 1, 4, and 7-weeks post occlusion. Tear cytokine concentrations were determined using an optimized CBA. Results. Acceptable assay performance measures were observed for the majority of cytokines, and sample attributes were favorable for tear CBA analysis. No between eye differences in tear clearance values were found prior to occlusion by the Standardized Visual Scale test (p = 0.23). Following occlusion, reduced tear clearance was observed for the occluded eye at week 1 (p = 0.02), week 4 (p = 0.01), and week 7 (p < 0.0001). For the tear cytokines assayed, no statistically significant differences were observed, nor was there a statistically significant interaction with time. Conclusion. Tear cytokines can be reliably measured by CBA using an optimized protocol. Concentrations remain stable during periods consistent with sample collection. Unilateral punctal occlusion reduces tear clearance when compared to the fellow non-occluded eye in normal subjects. However, this does not alter the concentration of tear cytokines compared to pre-occlusion levels or when compared to the fellow non-occluded eyes. |
