Usability of different tear fluid collection methods to analyze ocular surface protein, lipid, and cells

Tear fluid (TF) collection enables non-invasive ocular surface investigations. Tear wash (TW), microcapillary tube (MC), and Schirmer strip (SS) methods are commonly used; each with its strengths and limitations. Comparing the outcomes is dependent on the research goals. The purpose of this study is to compare protein and lipid recovery among the three techniques and compare white blood cell (WBC) differentiation between automated and manual methods.

TF samples were collected from 10 participants using TW, MC, and SS. Total protein and lipid content were determined using infrared spectrometry (Direct Detect, Millipore Sigma). TW samples were collected from 9 other participants and WBC differentiation was compared between automated hematology (DxH 500, Beckmann Coulter), manual hemocytometry (HEMO), and H&E staining. Mixed-effects analysis, Bland-Altman, paired t-tests, or the non-parametric equivalents were used.

Total protein content was the greatest for SS samples (471 ± 222 μg) and lowest for MC (88 ± 33 μg), (P = 0.0004) with the TW method (275 ± 215 μg) in between. Total lipid content was highest in TW (2.31 ± 0.56 Absorbance units (AU)) samples compared to MC (0.04 ± 0.02 AU) and SS (0.50 ± 0.19 AU), (P < 0.001). For the second analysis, total WBC count (median; IQR) was greater with the DxH (603 cells/µL; 327, 2365) compared to manual HEMO (475 cells/µL; 265, 2067), (P = 0.004), with a BA bias of -209 ± 209 cells/µL (LOA: -620 to 202 cells/µL). The coefficient of variation was significantly lower for the DxH compared to HEMO, P = 0.02.

These findings show that TF protein and lipid yields, along with cellular analysis, differ among TF collection methods and should be considered when designing TF studies. SS yielded the most protein and TW allowed cellular analysis. Automated hematology (DxH) provided repeatable total WBC counts.