PURPOSE: This paper examines the tear concentration of cystatin S (CST4), 
calcyclin (S100A6), calgranulin A (S100A8), and matrix metalloproteinase 9 
(MMP9), and the correlation between biomarker expression, clinical parameters, 
and disease severity in patients suffering from dry eye (DE). A comparison of 
the results is obtained via ELISA tests and customized antibody microarrays for 
protein quantification.
METHODS: This single-center, observational study recruited 59 participants (45 
DE and 14 controls). Clinical evaluation included an Ocular Surface Disease 
Index (OSDI) questionnaire, a tear osmolarity (OSM) test, the Schirmer test 
(SCH), tear breakup time (TBUT), fluorescein (FLUO) and lissamine green (LG) 
corneal staining, and meibomian gland evaluation (MGE). Tear concentrations of 
CST4, S100A6, S100A8, and MMP9 were measured using standard individual ELISA 
assays. The levels of CST4, S100A6, and MMP9 were also measured using customized 
multiplexed antibody microarrays. Correlations between variables were evaluated, 
and a significance level was p value <0.05.
RESULTS: The quantification of tear protein biomarkers with ELISA showed that 
the concentration of CST4 was significantly (2.14-fold) reduced in tears of DE 
patients in comparison with control (CT) subjects (p < 0.001). S100A6 and S100A8 
concentrations were significantly higher in the tears of DE patients (1.36- and 
2.29-fold; p < 0.001 and 0.025, respectively) in comparison with CT. The MMP9 
level was also higher in DE patients (5.83-fold), but not significantly (p = 
0.22). The changes in CST4 and S100A6 concentrations were significantly 
correlated with dry eye disease (DED) severity. Quantification of CST4, S100A6, 
and MMP9, using antibody microarrays, confirmed the ELISA results. Similar 
trends were observed: 1.83-fold reduction for CST4 (p value 0.01), 8.63-fold 
increase for S100A6 (p value <0.001) and 9.67-fold increase for MMP9 (p value 
0.94), but with higher sensitivity. The biomarker concentrations were 
significantly associated with the signs and symptoms related with DED.
CONCLUSIONS: S100A6, S100A8, and CST4 diagnostic biomarkers strongly correlate 
with DED clinical parameters. S100A6 and CST4 are also useful for grading DE 
severity. The multiplexed antibody microarray technique, used here for tear 
multi-marker quantification, appears more sensitive than standard ELISA tests.