Survey of cytokines in normal canine tears by multiplex analysis: A pilot study.

Objectives: The purpose of this pilot study was to use a multiplexed assay to measure cytokines in normal stimulated canine tears.

Methods: 25 healthy dogs were included in the study. Stimulated tears were collected in capillary tubes from the right (OD) and left (OS) eyes and stored at -80 °C until batch sample analysis was performed. The samples were analyzed utilizing Luminex canine-validated multiplex beads on a Bio-Rad multiplex analyzer for IL-2, IL-6, IL-7, IL-8, IL-10, TNF-α, and IFN-γ. Based upon previous human studies, tears were initially evaluated at a 1:10 dilution. Eight random samples were later re-analyzed without dilution.

Results: Diluting the samples 1:10 rendered all analytes undetectable except IL-8. A repeat analysis of eight randomly selected undiluted samples still demonstrated very low cytokine levels except for IL-8 (16/16 eyes; 2254 ± 1677 pg/ml OD, 1095 ± 786.8 pg/ml OS); and IFN-γ (15/16 eyes; 13.37 ± 13.08 pg/ml OD,16.08 ± 19.4 pg/ml OS).

Conclusion: This pilot study is the first to analyze cytokines in canine tears. This study demonstrated that IL-8 is consistently detected in both diluted and undiluted samples, but undiluted samples may be superior to 1:10 diluted samples for evaluation of other cytokines in canine tears.