“I'm not leaving this office until you run an antinuclear antibody [ANA] test and give me a satisfactory explanation for why I'm not really sick," Caitlin Flynn told her doctor, wiping away tears of frustration and distress.
It had been five years since she experienced her first symptom and after years of desperately trying to get a diagnosis for the increasingly debilitating symptoms, she was at her wit's end. “I couldn't take it anymore,” she said, of both the untreated illness and the parade of doctors who had dismissed her symptoms. When the fifth primary care doctor Caitlin saw finally ran the ANA test and gave her the positive result, confirming that she had systemic lupus erythematosus (SLE, or lupus for short), she was finally referred to an autoimmune specialist.
Lupus is a disease that is notoriously difficult to diagnose because the symptoms can be vague, differ from person to person, mimic other diseases, and come and go over time. It is a chronic, autoimmune disease, where a person’s immune system malfunctions and turns on its own body, attacking healthy tissues and organs. Multiple organ systems can be affected, including the joints, skin, kidneys, blood cells, brain, heart, and lungs. As no two cases of lupus are alike, it is not unusual for it to take several years for patients to be diagnosed. Evidence suggests that the earlier a person is diagnosed and treated, the lower the chances of irreversible organ damage and death.
Can precision medicine improve the chances of people like Caitlin getting diagnosed and treated sooner for lupus or other autoimmune diseases? The answer is yes.
Lupus is one of several diseases that Dr. Lei Zhou and his team at the Singapore Eye Research Institute (SERI) are working on. They are discovering clinical biomarkers that will help diagnose these diseases using the proteomic and metabolomic analysis of tear fluid samples.
One of the key technologies these researchers are employing is mass spectrometry. Proteomic and metabolomic experiments are performed on advanced analytical instruments such as ultra-high performance liquid chromatography (UPLC) systems, nano-flow LC, and time-of-flight (TOF) mass spectrometers designed for the collection of large amounts of highly precise quantitative and qualitative proteomic and metabolomic data.
The research team has applied these technologies to the analysis of tear fluid samples. Tears are made up of three layers, comprised of lipid, aqueous, and mucin components. All three layers are analyzed and the profile of tear proteins quantified and characterized for many diseases. To date, the research team has banked almost 4,000 tear proteins and a few hundred small molecule metabolites in their database. They have investigated 15–20 ocular diseases, as well as several systemic diseases that can affect the eyes, including lupus (a collaborative effort between Zhou, a proteomics scientist, and rheumatologist Dr. Julian Thumboo at Singapore General Hospital). Being able to determine as early as possible when a disease flare has started is important for the timely administration of treatment to optimize patient outcomes, including minimizing tissue damage and complications from lupus.
Building a point-of-care test
Using LC-MS/MS with isobaric tags for relative and absolute quantitation (iTRAQ), thousands of proteins are labeled and quantified at the same time from patient tear fluid samples. The iTRAQ technique provides high accuracy and sensitivity, and so is widely used for protein separation and analysis. In this way, specific proteins in tear fluids can be discovered that distinguish between patients with lupus who have a flare of their condition and those who are in remission. Once the candidate biomarkers have been discovered using LC-MS/MS with iTRAQ, high resolution multiple reaction monitoring (MRMHR) MS scans are used to identify which candidates are analytically valid. The MRMHR analysis has the power to detect a broad dynamic range of proteins with both very high specificity and sensitivity. The analytically validated candidate biomarkers are then tested in a prototype point-of-care test (POCT) on real-world tear fluid samples from a cohort of patients with lupus. These POCTs based on tear fluid samples provide an indicative view of the body’s health status; with their help, the eyes could truly serve as a “window to the soul.”
The prototype POCT is being developed in collaboration with nanotechnology scientists at another institution. It is based on a simple method that will enable patients in remission to quickly test themselves at home using their tears. The early detection of a flare will enable the patient to notify their doctor and seek treatment.
Current methods for monitoring lupus disease activity are invasive and involve antibody-based enzyme-linked immunosorbent assays (ELISAs) that bind to specific proteins in blood samples. A home test kit would be much more user-friendly and could allow for more frequent testing to better monitor for flares. Non-invasive testing offers much better quality of life and home testing allows people to take control of their health, empowering individuals with chronic conditions with the ability to better predict and thus cope with their flares.
The next 10 years
Advanced analytical science is key for the discovery of biomarkers, which will go on to be used in routine clinical tests. The quantitative profiling of tear proteins to create non-invasive tests for autoimmune disease is a novel approach to biomarker-driven diagnostics and chronic disease management. The research of Zhou and his colleagues at SERI is just one example of how mass spectrometry and other rapidly advancing technologies are enabling scientists to realize a vision of precision medicine and personalized health for all of us. The advances in MS, with tools such as SWATH for data-independent acquisition (DIA), for comprehensive high-throughput proteomic profiling will enable scientists to discover more and more markers for changing health status and disease.
Within the next 10 years, we will see tests not only for the diagnosis of diseases like lupus, which are difficult to detect, but also for a whole host of other disorders and health conditions. Many diseases are multifactorial, and many health conditions have a preclinical stage, where intervention has the strongest chance of halting, preventing, or even reversing pathology. Home testing will become more commonly prescribed by physicians and also more readily available as kits direct to consumers. Similar to direct-to-consumer genetic tests and ketone urinalysis strips, proteomic and metabolomic panels will be accessible over the counter to allow individuals to test their tears and thus monitor for any important changes in their protein and metabolic make-up on a regular basis. This can provide information on not only a single disease but on multiple aspects of our physiology, because tear proteins and metabolites change in response to alterations or damage in other parts of the body.
Personal health monitoring and biomarker testing is already being embraced by many individuals, empowering them with the data and knowledge to take proactive steps to maintain and optimize their health. People are using this information to decide on what to eat, when to exercise, and when to see a doctor. In the next decade, such personal testing and proactive health monitoring will become even more commonplace. People will have greater opportunities to seize control of their health and improve their chances of avoiding illness. And should someone start to fall ill, she or he could detect it earlier and thus be better able to seek treatment in time for a good outcome. In any case, these tools will help us access information to realize a healthier future.