The Impact of Post-Tear Collection Storage on Tear Film Substance P Concentration

Abstract

Purpose

Substance P is a sensory neuropeptide increasingly used as a biomarker for ocular and systemic neuropathic conditions. Due to the limited studies on tear storage conditions compared to other bodily fluids including blood and urine, the aim of this study was to investigate whether different storage durations at 4 °C can impact on substance P concentrations prior to storage at −80 °C. This is important to assess potential practical limitations in the handling and storage of tear fluid essential.

Methods

Tears were collected and pooled from both eyes of 31 healthy participants using the flush tears method. The samples were centrifuged and aliquoted into three sets of microcentrifuge tubes with each stored at 4 °C for <2 h, 4 h or 6 h (Timepoints 1, 2 or 3). After each respective storage duration, the aliquoted samples were than stored at −80 °C before analysis, within 6 months. Tears were analyzed for the concentration of substance P and the total protein content (TPC).

Results

Substance P concentrations across the three timepoints were not significantly different (p > 0.05), including Timepoint 1 (Median [interquartile range]: 10.7 ng/ml [1.6-37.9]), Timepoint 2 (10.9 ng/ml [1.6-32.6]) and Timepoint 3 (5.2 ng/ml [1.3-25.2]). There were also no significant differences in TPC concentrations measured at the three timepoints, including Timepoint 1 (3.1 mg/ml [1.7-3.8]), Timepoint 2 (2.9 mg/ml [1.9-4.1]) and Timepoint 3 (2.7 mg/ml [1.6-3.7]).

Conclusions

While the levels of substance P were stable while stored at 4 °C prior to proper −80 °C storage and analysis, future research should investigate the impact of other storage conditions such as ambient room temperature to optimize the feasibility of using tears for biomarker purposes in clinical settings.

Keywords:

• Biomarkers
• neuropeptides
• proteins
• substance P
• tears

Acknowledgements

We would like to thank Dr Thomas John Naduvilath for his statistical guidance for this manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data that support the findings of this study are available from the corresponding author (JCBC) upon reasonable request.

Additional information

Funding

JCBC is supported by an Australian Government Research Training Program Scholarship.