Flow Cytometry Could Offer New Insights into Complex Eye Disease

A new flow cytometer in Duke’s Department of Ophthalmology is giving researchers a new way of looking at ocular tissue samples and the unique biomarkers associated with certain diseases—a view that will someday lead to a diagnostic tool for complex eye diseases and better insights into treatment strategies.

Using the most advanced technology currently available, Duke’s flow cytometer processes ocular tissue and characterizes cells based on specific signatures.

“With data from the cytometer, we are beginning to ask if these cell signatures can actually predict disease outcomes and whether they can be used in precision medicine,” explains Daniel R. Saban, PhD, a Duke associate professor of ophthalmology who was instrumental in obtaining the equipment.

“In clinical practice, when evaluating patients who have chronic uveitis, it can sometimes be challenging to determine the exact etiology of the underlying inflammation—whether it is autoimmune, infectious, or possibly a malignancy like lymphoma masquerading as chronic inflammation,” says Dilraj Grewal, MD, a Duke ophthalmologist and retinal surgeon. “Routine blood-based testing only gives us an answer in about half the patients. In the rest, we often have to obtain a sample from the fluid in the front of the eye (aqueous) or the back of the eye (vitreous). Analysis of this fluid can give us more information on the disease; however, current methods of analysis may still not give us an answer in almost half the cases. This is a source of frustration both for the patient and the physician.”

The basic principle of flow cytometry is the passage of cells in single file in front of a laser so they can be detected, counted, and sorted. Cell components are fluorescently labeled and then excited by the laser to emit light at varying wavelengths.

Saban explains that each cell marker is positive or negative, and each generates two levels of data. A traditional flow cytometer searches for 10 markers simultaneously; however, Duke’s state-of-the-art equipment processes at least 36 at a time, which results in a rich data set that requires artificial intelligence to analyze.

The equipment arrived at Duke shortly before the COVID-19 pandemic began and was quickly deployed to process blood samples from patients who were tested for the virus to analyze the immune system response in these patients. “We had access to a large repository of samples from asymptomatic patients, some who had mild to severe disease, and some with critical disease. We were able to look at changes at the single cell protein level with the flow cytometer,” says Saban. (See study: Mucosal Associated Invariant T (MAIT) Cell Responses Differ by Sex in COVID-19)

Several studies based on these analyses are currently underway, and the results could lead to answers about how viruses like COVID-19 manifest in the body, why men seem to be more susceptible to the novel coronavirus than women, and other unanswered questions.

“This new generation multi-parameter flow cytometry is a very exciting technology that might allow us to determine unique cytometric profiles for different eye diseases,” says Grewal. “Accurate identification of such signatures is critical to be able to deliver the correct treatment in a timely manner, whether it is immunosuppression in cases of autoimmune inflammation, antimicrobial treatment in cases of infections, or chemotherapy in cases of lymphoma.”

Although the flow cytometer is primarily being used in a research setting only and on a limited basis in the clinics, Saban believes it will soon play a central role in making clinical decisions and offer a number of benefits for improving patient care, including:

• Diagnostic capabilities with improved speed and accuracy for identifying complex diseases
• The ability to stratify patients according to disease severity and trajectory to facilitate more efficient drug trials
• Improved insights for developing treatment strategies based on early knowledge of whether a patient will benefit from certain therapies
• The ability to detect patterns in blood analyses that humans are unable to see, providing additional information to make accurate diagnoses

“We often see patients who have had a delay in diagnosis of their chronic inflammation for several months or even several years and such persistent inflammation can permanently impact the vision by causing damage to the retina and other parts of the eye,” says Grewal. “Achieving early and accurate diagnosis from eye fluids with this technology carries the potential to be a huge advance in the care of patients with uveitis by allowing us to deliver timely treatment.”