Image: Nanosight NS300 can visualize and measure suspended particles in terms of size, light scattering intensity, fluorescence, and count (courtesy of the Mark Wainwright Analytical Center).
Current clinical studies face significant challenges in isolating disease subtypes with precise molecular signatures and in 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 the accuracy of diagnosis.
Since small extracellular vesicles (SEVs, exosomes) appear to have specific roles in many biological processes, including immune regulation, angiogenesis, tumor invasion and cell migration, exosome-based liquid biopsy techniques have made exosome-based liquid biopsy techniques an attractive option for disease classification and prognosis. has offered. Tears are suitable for sample collection because fluid can be collected quickly and non-invasively.
Medical scientists from Wenzhou Medical University (Wenzhou, China) and their colleagues conducted a retrospective case–control study involving patients with dry eye and type 2 diabetes. A tear sample was collected by placing a Schirmer paper in each eye, and the wet length was recorded at or after 5 min of collection (length reached 30 mm at 5 min).
The tear mixture was removed from the Schirmer strip by shaking at 4 °C for 30 min and centrifuging in two steps (200 g for 10 min and 3,000 g for 10 min) to remove cells and other impurities. Subsequently, exosomes were purified with a rapid isolation system under a negative pressure of 40 kPa and a conversion time of 30 sec, respectively. Samples were loaded onto balanced size exclusion chromatography columns (original qEV, IZON Science Ltd., Oxford, UK) and 16 sequential 0.5 ml fractions were separated by addition of PBS. Fractions 8–10 were pooled and concentrated to a final volume of 150 μL. Nanoparticle tracking analysis (NTA) was performed on a NanoSight NS300 system (Malvern Panalytic, Malvern, UK). Other methods included in the study included western blot analysis, on-device exosome detection, proteomic analysis, and exosome miRNA sequencing.
Based on proteomic evaluation of the extracted proteins, the researchers were able to differentiate between healthy controls and patients with different types of dry eyes. Similarly, Tear Embedded Exosome Analysis via Rapid Isolation System (iTEARS) allowed the team to observe differences in microRNAs in patients with diabetic retinopathy and without eye conditions, suggesting that the system can track disease progression. can help to. The team says this work could lead to a more sensitive, faster and less invasive molecular diagnosis of various diseases, using only tears.
The authors concluded that they established iTEARS to understand disease biomarkers in a tear, 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. Applicable in a wide range of disease types and validated with abundant clinical cases, they anticipate that their iTEARS tear test will be an alternative tool to point-of-care testing. The study was published July 20, 2022 in the Journal. was published in ACS Nano
Wenzhou Medical University
IZON SCIENCE LIMITED