Human tear analysis with miniaturized multiplex cytokine assay on “wall-less” 96-well plate

PURPOSE Tears are a particularly limited body fluid and commonly used in the diagnosis of patients who have ocular diseases. A popular method for analysis of ocular inflammation in tears uses Luminex® bead multiplex technology to generate valuable multiple cytokine profile outputs with 25-50 µl tear sample volume. We propose a method for measuring tear cytokines with 5 μl tear sample volume and 80% reduced Luminex reagents compared to previous protocols. METHODS Using human tears pooled from 1,000 participants, the DA-Bead-based method running at 5-20 µl volume, using manual pipetting, in conjunction with a magnetic Luminex cytokine (four-plex) panel assay in a 96-well format was performed and validated for tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-1β, and IL-6. RESULTS Upon use of the DA-Bead method at the 5 μl volume with cytokine standards, the concentrations of each of the four cytokines were found to be linear over a range of 3.5-4 log pg/ml with an intra-assay coefficient of variation (CV) ≤5%, inter-assay %CV ≤10%, and accuracy within the 70-130% range. Upon use of a 5 µl healthy pooled tear sample, cytokine concentrations were detected with a precision intra-assay %CV ˂ 20% for IL-6, IFN-γ, or TNF-α or 30.37% with IL-1β. The inter-assay %CV with tears was ≤20.84% for all cytokines. Tear volumes run at 5 μl on DA-Bead produced a similar cytokine expression profile at a 1-month interval and were highly correlated with the larger 10 μl-based tear sample volume cytokine profile with R(2) = 0.98. CONCLUSIONS DA-Bead assay is highly sensitive and reproducible and has a performance profile that is potentially suitable for use in standard clinical scenarios. Considering the use of as little as 5 µl of assay beads and 5 µl sample, this is also likely to reduce the assay cost significantly and ease diagnosis of patients with ocular diseases.