Diagnosing disease by isolating biomarkers in tears – Technology

Image: The NanoSight NS300 can visualize and measure suspended particles in terms of size, light scattering intensity, fluorescence, and number (Photo courtesy of Mark Wainwright Analytical Center).

Current clinical studies pose significant challenges in differentiating disease subtypes with precise molecular markers and tracking disease progression in a non-invasive manner. Identifying molecular clues in patient samples, such as specific proteins or genes in vesicular structures called exosomes, can improve diagnostic accuracy.

Since small extracellular vesicles (sEVs, exosomes) reveal specific roles in several biological processes, including immune regulation, angiogenesis, tumor invasion, and cell migration, exosome-based liquid biopsy technology has offered an attractive alternative for disease classification and prognosis prediction. Tears are suitable for sampling because fluid can be collected quickly and non-invasively.

Medical scientists at Wenzhou Medical University (Wenzhou, China) and colleagues conducted a retrospective case-control study that included patients with dry eye and type 2 diabetes. Tear samples were collected by placing Schirmer paper in each eye, and the moistened length was recorded after 5 minutes. minutes of collection or after completion (length reaches 30 mm in 5 minutes).

The tear mixture was eluted from the Schirmer strip by shaking at 4 °C for 30 min and centrifuged in two steps (200 g for 10 min and 3,000 g for 10 min) to remove cells and other impurities. Subsequently, the exosomes were purified by a rapid isolation system under a negative pressure of 40 kPa and a conversion time of 30 s each. Samples were loaded onto a balanced size exclusion chromatography column (original qEV, IZON Science Ltd., Oxford, UK) and 16 successive 0.5 ml fractions were eluted with the addition of PBS. The eluted fractions 8-10 were collected and concentrated to a final volume of 150 L. Nanoparticle Tracking Analysis (NTA) was performed on the NanoSight NS300 system (Malvern Panalytical, Malvern, UK). Other methods included in this study included Western blot analysis, on-device exosome detection, proteomic analysis, and exosome miRNA sequencing.

The investigators were able to differentiate between healthy controls and patients with different types of dry eye based on the proteomic evaluation of the extracted protein. Similarly, Tear Embedded Exosomes Analysis via the Rapid Isolation System (iTEARS) allowed the team to observe microRNA differences between patients with diabetic retinopathy and those without the eye condition, suggesting that the system could help track disease progression. The team says this work could lead to more sensitive, faster and less invasive molecular diagnosis of various diseases, using only tears.

The authors conclude that they established iTEARS to decipher disease biomarkers in tears, revealing the promising role of tear exosomes in disease classification and tracking the course of eye disorders and other diseases, such as neurodegenerative diseases and cancer. Applied across a wide range of diseases and validated with many clinical cases, they anticipate that their iTEARS tear test will become an alternative tool for on-site testing. The study was published on July 20, 2022 in the journal DHW Nano

Related links:

Wenzhou Medical University

IZON Science Ltd.

Malvern Analytic

Roderick Gilbert

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