Abstract
Purpose :
To evaluate the effect of pre-analytical sample handling on tear fluid protein levels.
We empirically tested variations of common tear collection and handling procedures.
Methods :
We created sample sets that address the effect of tear collection, storage and extraction on protein recovery. Tear fluid was collected using Schirmer’s strips from healthy subjects. Proteins were extracted by elution and centrifugation. The total protein content was measured with the BCA assay.
Results :
Protein recovery was the highest after direct (‘fresh’) extraction. Minimal protein loss was found after intermediate storage at -80°C and -20°C (-3.1% and -4.5%, respectively). Protein concentration in the ‘wet’ storage condition of strips was 8.5 ± 6.0% higher compared to ‘dry’ storage. Protein recovery increased with increasing extraction volumes but also results in higher dilution of the sample. Sonication did not improve protein extraction efficiency. Protein concentration within the Schirmer’s strip was the highest in the 0-5 (+head) mm region (1034.7 ± 72.7 μg/mL). Consecutive tear fluid collection did not greatly affect protein concentration. Protein recovery decreased with protein concentration but was independent of molecular weight.
Conclusions :
There is a need to construct standardized operation procedures for pre-analytical handing of tear fluid in order to facilitate to build tear fluid biobanks and the use of tear fluid-based biomarkers in the clinical setting.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.