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Harvard, Wenzhou Medical University Researchers Develop Method for Tear-Based Dx


NEW YORK – A team of researchers from Harvard Medical School and China's Wenzhou Medical University are making strides toward using tears to diagnose a range of different diseases.

The method — incorporated tear-exosomes analysis via the rapid-isolation system, or iTEARS — and its applicability to dry eye disease and diabetic retinopathy was laid out in a paper published last month in ACS Nano. iTears draws on previous work from the research team published in Nature Methods last year explaining the development of Exodus — an exosome detection method via the ultrafast-isolation system. iTears applies the exosome purification and enrichment methods of Exodus to tear exosomes and was developed to see if health conditions could be determined using tears, said Luke Lee, a professor of medicine at Harvard Medical School and an author on both papers. 

Within tears, there are biological nanoparticles shed by cells — exosomes — that contain proteins, lipids, RNA, and other biomarkers that can indicate certain diseases. According to Lee, exosomes "provide us very crucial information on pathogenesis or pathological processes," but they are "tiny, so [they are] not easy to collect." They're "very delicate" and must be treated "very gently."

Tears collect exosomes from a variety of different organs and tissues throughout the body, making them a beneficial sample type for detecting various diseases, he said. Because tears are a "more pure liquid" than other bodily fluids, using tears is "much cleaner" than saliva or nasal samples. Fei Liu, principal investigator at the school of ophthalmology and optometry at Wenzhou Medical University and another author on the papers, added via email that tears have "low potential contaminant interference" compared to other biological fluids.

The researchers have tried using their technology with multiple bodily fluids, including urine, blood, and saliva, but those samples are harder to purify and more time-consuming to work with, Lee said. For example, saliva is "very viscous" and contains a lot of other material that needs to be separated out before exosomes can be analyzed, he added. The separation and purification steps are "more challenging processes," he said.

Using tears for biomarker analysis isn't new since they are "highly accessible," making it easier for researchers to develop noninvasive methods to uncover molecular signatures of disease, Tony Jun Huang, a professor of mechanical engineering and materials science at Duke University, said via email. 

He added that researchers have found that tear exosomes have an abundance of immune response-related proteins and miRNA which can be applied to the diagnoses of multiple diseases, including glaucoma, multiple sclerosis syndrome, and cancers noninvasively.

Since Lee and his colleagues decided to use tears as the sample type, the team selected eye-based diseases for the technology's first applications — dry eye disease and diabetic retinopathy, Lee said.

But while other researchers have used tear exosomes for diagnosis, the sample preparation method developed by Lee and Liu's team is unique.

Isolating the exosomes and removing other material from the tears via this system requires a nanoporous membrane, a transducer to generate acoustic waves, and the sample itself. The tear sample is placed on top of the membrane with the transducer. Electrodes are attached on either side of the membrane, controlled via the transducer. Once activated, the transducer causes the membrane to vibrate and bounce the sample back and forth, and the resonant harmonic oscillation of the membrane causes the exosomes to quickly separate from the rest of the material in the sample and be collected underneath the membrane.

Once the exosomes have been isolated and purified, they can be processed using either mass spectrometry or RNA sequencing. The team is currently working to discover biomarkers related to different diseases, but both Liu and Lee said once some of those markers are confirmed, mass spec or sequencing wouldn't be necessary. Instead, an immunoassay or photonic PCR chip could be used to make a rapid molecular diagnosis using the validated biomarkers.

Right now, the researchers are focusing on accumulating and identifying selective biomarkers from tears, but future plans involve developing a more "portable system" to allow the technology to be used clinically at the point of care, Lee said.

One issue when using tear exosomes for diagnosis, Duke's Jun Huang noted, is that ultra-centrifugation and size-exclusion chromatography — considered the gold standard methods for exosome separation — have limited use clinically because of low recovery rate, an hourslong processing time, and large sample consumption.

iTears, however, shows a higher recovery rate and faster processing time than those traditional methods, demonstrating its potential applicability in clinical settings, he said. The isolation and purification method takes about five minutes and requires only "a few teardrops" — about 10 microliters of fluid, the researchers wrote in the ACS Nano paper.

In the ACS Nano study, the researchers detected 426 proteins from tear exosomes related to dry eye disease and demonstrated three that can be used to classify subtypes of dry eye disease. They also investigated 484 microRNAs in tear exosomes, showing that four are dysregulated during diabetic retinopathy development.

Lee and Liu said one of their dreams is to move the technology into the home for disease monitoring, although that dream is a long way off. He noted that it will likely take the team a few years to collect and study other disease biomarkers, but that a more user-friendly system to adapt the technology for clinical use could be developed in parallel with the biomarker discovery to get it commercialized faster.

The researchers may also draw on other published works, using the technology to determine if previously discovered biomarkers can be found in tears and then developing a test using those preexisting biomarkers, he said.

Another goal is to study the molecular expressions in exosomes from tears related to emotional stress, he said. 

Lee and his team's work has implications for the rest of the diagnostics field, too. Duke's Jun Huang said that "incorporating downstream extracellular vesical analysis with extracellular vesical separation technology will be extremely important for expanding the field of disease diagnostics."

However, he noted that he'd like to see if the technology could be "implemented for exosome isolation with other biofluid sample types, such as sweat, urine, and blood," which would "not only benefit our research community but also make a huge impact in disease diagnostics."