Purchase this article with an account.
R. J. Fullard, L. E. Kehinde; Do Tear Proteins Interfere with Tear Cytokine Cytometric Bead-based Assays (CBAs)?. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2549.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Tear cytokine profiles can yield important information about ocular surface health. In a previous study of 27 tear cytokines in normal patients, stimulated (S) tear levels were higher than non-stimulated (NS) in many cases. This difference demonstrates the importance of tear stimulus control. However, the counterintuitive trend with stimulus suggests the possibility of assay interference by endogeneous tear components. This should be investigated in both sample types.
NS tear samples and paired S samples (nasal stimulus, sample preceded by 20 µL washout) were collected atraumatically by polished micropipette from normal patients. Pooled and non-pooled aliquots of each sample type were stored at -80°C in assay buffer. Interference effects were investigated by measuring cytokine recovery in standards spiked separately into NS tear-equivalent levels of purified sIgA, lactoferrin, or lysozyme. NS tear levels of purified sIgA were then spiked into pooled and non-pooled NS and S tear samples. The effect of adding copper sulfate, known to decrease endogeneous lysozyme interference in susceptible assays, was also tested. Cytometric bead-based assays of all samples were run on a Luminex 200 system using a Bio-Rad 27-Plex kit.
Copper sulfate produced no significant change in standard or tear sample spike recoveries. For standard spike recoveries, lysozyme produced the greatest interference effect for 6 of the 27 cytokines, lactoferrin for 7, and sIgA for the remaining 14. IL-4, IL-10, IL-15, IP-10 and MCP-1 showed the greatest average departure from 100% recovery for the three standard spiking conditions. Lactoferrin spiked standards departed from 100% recovery by less than 10% for 13 cytokines, lysozyme for 16 and sIgA for 9 cytokines.
sIgA produces a greater interference effect than lactoferrin or lysozyme on spike-recovery of cytokines from standards and from tear samples. This may help explain why many measured cytokine levels are lower in NS tears, because endogeneous sIgA levels are higher. Additional studies are required to fully elucidate the sources of interference in tear CBA.
This PDF is available to Subscribers Only