Simultaneous measurement of six cytokines in a single sample of human tears using microparticle-based flow cytometry, allergies vs. non-allergies

Tears play an essential role in maintaining corneal and conjunctival integr ity by providing a tightly regulated, optimal extracellular environment cri tical to its numerous functions, which include anti-microbial defense, woun d healing and inflammatory responses such as allergies. Elevated levels of inflammatory cytokines have been reported in tears from various ocular dise ase states. Characterization of tear cytokines has been limited by the smal l volume (microliter amounts) attainable. This limitation was addressed wit h the newly developed Becton Dickinson Cytometric Bead Array (CBA), which c ombines the principles of the "sandwich" immunoassay with the capability of flow cytometry for simultaneous measurement of the characteristics of mult iple particles. This technique allows determination of six human cytokine ( IFN gamma, TNF alpha, IL-2, IL-4, IL-5, IL-10) concentrations simultaneousl y in a single tear sample. Tears were collected from the inferior fornix of non-allergic (n = 7) and allergic (n = 9) donors. Each tear sample or cyto kine standard was incubated with a mixture of capture Ab-bead reagent and d etector Ab-phycoerythrin (PE) reagent, and analyzed using flow cytometry. A ll six cytokines were detectable in both non-allergic and allergic tears. T ears from allergic donors contained significantly less IL-10 (p = 0.035), a nd had significant increases in the ratios of TNF alpha /IFN gamma. IL-5/IF N gamma and IL-5/IL-10 (p = 0.0008, 0.0124 and 0.011, respectively). The sm all volume required (5-10 mul/test) by the Cytometric Bead Array allows mea surement of all six cytokines from a single collection of tears. This decre ases collection time, minimizing the confounding effect of stimulation on c ytokine concentration in tears, as well as allowing calculation of cytokine ratios. (C) 2001 Published by Elsevier Science B.V.