Inside the Diagnostics Industry: One Drop of Blood Going a Long Way to Diagnose Disease

It was just over a year ago that Theranos (Palo Alto, Calif.) set the laboratory industry abuzz by unveiling its microsample testing services at Walgreens locations. While Theranos’s rapid results and transparent, low-cost pricing is still potentially transformative for the entire health care system, interest in other microsample testing scenarios continues to gain momentum. Among the purported benefits of microsample testing is the ability to conduct more frequent monitoring, especially if drop-sized samples can be self-collected. This scenario not only eliminates the inconvenience of traveling to blood draw sites for the sick, the elderly, or those in underserved areas, but it also eliminates the barrier to testing imposed by the fear of needles. All of the companies DTET spoke to involved in microsample testing herald the benefits of more frequent monitoring and improved patient compliance. Spot On Sciences (Austin, Texas) believes it can transform testing through innovation in the collection and storage of biological fluid samples. What if patients didn’t need to worry about coming in for blood draws? What if with a finger stick they could mail in their own self-collected, two-drop sample? Spot On Sciences’ CEO Jeanette Hill, Ph.D., believes this is possible through some enhancements to the collection of dried blood spot (DBS). “The idea came from talking to my mother, who lives in a rural area,” Hill tells DTET. “It is really hard for her to travel and get to a site to get a blood draw.” While DBS technology is not new (it has been used for newborn screening for 50 years), it is not widely used outside of newborn screening due to challenges with collecting quality samples. Traditional DBS requires a multistep process, and samples could be degraded from moisture, contamination, and sample loss. “It is hard to get a good sample in the hospital, let alone in the wilds of our kitchens, so the HemaSpot device tries to make it foolproof,” Hill says. The company’s patented HemaSpot device improves sample quality, simplifies collection, and allows for stable sample storage at room temperature. It collects and dries two drops of blood within a protective, credit card-sized cartridge that can be immediately shipped. The moisture-tight cartridge has a tamper-resistant latch and can be labeled with a unique bar code for sample identification that can be read by standard readers such as smart phones or laboratory scanners. Smart phone-based bar code recognition applications can be used to alert a lab that the sample has been shipped along with patient information and a time and date-stamp for the sample. The company was founded in 2010, and Hill was awarded DARPA Small Business Innovation Research (SBIR) Program Phase I funding to improve access to medical testing for elderly, homebound, rural, and economically disadvantaged patients by allowing easy remote sample collection and shipment. The award allowed the company to prove collection and analysis of time-relevant blood samples for medical research in chronic disease and population studies was feasible. This summer, Spot On received a $750,000 DARPA SBIR Phase II Option Award to scale-up manufacturing and quantification methods, and produce field-use data. Hill says the need to better understand the effects of circadian rhythms on biomarkers is behind the recent DARPA award. A Spanish study has shown that the survival of at-risk patients is increased up to fivefold by simply taking blood pressure medications at bedtime instead of morning. The cause of this is likely due to circadian rhythms, but such studies have been difficult, given the need for phlebotomist collections in the early morning and late evening. Ramon Hermida, the chronobiology expert behind the Spanish study, will utilize HemaSpot to initiate large-scale, low-cost patient studies in blood pressure and hypertension next year. The HemaSpot device is designed to absorb a target volume of 80µL of blood. The fan-shaped, dried sample can be removed in portions (each blade is approximately 11.5 percent of the total fan, meaning that each blade can carry approximately 9µL to 10µL of blood per blade), enabling multiple tests to be run from a single self-collected sample. Markers that can be measured from the DBS include proteins, nucleic acids, and small molecules. “You can do almost all tests from HemaSpot samples,” Hill says. “With advances in mass spectronomy and polymerase chain reaction, you can do so much with two drops of blood. It works really well with these newer technologies.” The company sells directly to laboratories. Initial interest has been strong in the areas of HIV and infectious disease testing as well as with chronic conditions and wellness testing. The company is awaiting CE-mark for Europe and is working with the U.S. Food and Drug Administration. Development is also currently under way on devices for collection of additional fluids (urine and saliva) and tissue. ELISA-Based Nucleosome Testing for Cancer Another company reinvigorating an old-school technology is VolitionRx (Belgium). The company was founded in 2010 with the goal of making cancer diagnosis as simple as a blood test for cholesterol by bringing together the long-established enzyme-linked immunosorbent assay (ELISA) diagnostic technology with cutting-edge nucleosome detection and analysis techniques. The company believes this is possible using its Nucelosomics technology, which measures signatures of nucleosomes in circulating blood. Like a “beads-on-a-string” necklace, each bead or nucleosome is DNA wrapped around a core of histone proteins. As cells die, the string is naturally broken down with individual nucleosomes released into the blood. With the rapid cell turnover associated with cancer, nucleosome levels rise in cancer patients’ blood. “The basic idea is that remnants of chromosomes are circulating in blood and that the chromosomes of cancer patients are different than healthy patients’,” Jake Micallef, Ph.D., VolitionRx’s chief science officer, tells DTET. “We are extending that to look at histone variants as well.” The company’s NuQ assays can be run on multiple ELISA platforms, including manual plates, automated machines, or point-of-care configurations, which the company will pursue in “relatively short order.” NuQ assays have been shown to both distinguish caner patients from healthy ones or those with related, but noncancerous, conditions as well as distinguish different types of cancer. “The reason ELISA is very good for what we do is that it is ‘old hat’ and that every hospital has a machine that they can do one more application on,” Micallef says. The company is developing assays able to detect methylation in the DNA, methylation in the histones, as well as variants in histones, and nucleosome-protein complexes. The company’s first commercially available clinical application of the Nucelosomics technology will be in colorectal cancer (CRC) testing. CRC screening is plagued by compliance issues. Yet CRC screening remains a large market, 150 million people in Europe and 87 million in the United States, the company says. “We chose CRC in the first instance because it has the best gold standard. Yes, there is the greatest competition with Exact Sciences and EpiGenomics; nonetheless, because there is competition shows there is a market and there is a gold standard to compare the assay to,” Micallef says. By comparison, with lung cancer, he says, if the assay is positive in an apparently healthy person, it could be 10 years to 15 years before symptoms develop, and there is no other diagnostic for comparison. The company is currently conducting analysis of its 4,800-subject CRC trial. However, they recently reported encouraging top-line results from an initial group of 938 subjects, which shows the NuQ test accurately detected colorectal cancer in 84 percent of subjects (78 percent specificity) and detected 60 percent of adenomas (polyps). The assay performed with similar accuracy for the detection of both early (I or II) and late-stage (III or IV) disease and was able to differentiate CRC from other gastrointestinal conditions. The company will seek CE mark in Europe next year and will start a pivotal trial in the United States next year. “With small samples, you move to pin pricks, which can enable tests to be performed more often. You can almost take patient compliance out of the equation,” Micallef says. “At the moment [in Europe] only 50 percent do fecal screenings. With small blood samples you move from actively opting into a nasty test to a situation where it can almost be done automatically, like cholesterol tests, liver enzymes, and cancer screening.” The company also recently released pilot study results of the NuQ platform’s ability to detect lung cancer in both blood and sputum. Both sample sources could detect early- and late-stage lung cancer with high sensitivity and specificity and could differentiate lung cancer from chronic obstructive pulmonary disease, independent of smoking status and age. This was the first trial of nonblood samples on the NuQ platform, “greatly extending [Nucleosomics technology’s] potential applications,” the company says. The company has other ongoing trials in colorectal cancer, lung cancer, prostate cancer, and endometriosis. An Immunosignature A single drop of blood also holds an immunosignature that can provide clues about the body’s disease states. This approach avoids the need to measure the pathogen directly and capitalizes on the body’s own immune-related amplification processes to ensure sensitive results, while using miniscule sample sizes. HealthTell’s (San Ramon, Calif.) Immunosignature technology is applicable across over 30 diseases, including neurological, autoimmune, oncologic, metabolic, and infectious diseases. It was developed as “an alternative to the typical reductionist biomarker paradigm” and leverages the response of antibodies to disease-related changes, as well as the inherent signal amplification associated with antigen-stimulated B-cell proliferation. “It is simultaneous detection and identification of multiple diseases with a single assay that underlies the true potential of this approach as a disruptive force in healthcare,” writes Stephen Albert Johnston, Ph.D., from Arizona State University (Tempe), whose work provided the foundation for HealthTell, in a July 14 Proceedings of the National Academy of Sciences paper. “This, combined with the fact that serum antibodies are robust to handling such that a drop of blood can be sent dried on filter paper through the mail, should enable frequent, inexpensive monitoring for many different diseases.” The company says it can provide a real-time measurement of an individual’s health. The platform is based on a high-density peptide array. The antibodies in diluted blood are incubated with a microarray of thousands of random sequence peptides. The pattern of binding to these peptides is the immunosignature. “An important aspect of this approach is that it senses essentially all antibodies raised to the disease and detects each of the antibodies as separable binding patterns composed of unique molecular recognition elements,” Johnston says. “This differs from, for example, an ELISA, which might sum the contributions of many different antibodies using a single protein, cell, or virus capsid.” Johnston also says that by highly diluting the blood it improves sensitivity and specificity, prevents other blood proteins from significantly binding to the arrays, and ensures the assay is sample-sparing. The fabrication of these arrays leverages equipment and processes similar to those used in the manufacturing of silicon-based electronics, making it both cost-effective and scalable, unlike printed microarrays. The company has currently demonstrated success using 10 million peptides per slide (330,000 peptides per assay) on silicon wafers. This October, the company announced $13.5 million of funding to help expand the development and commercialization. Takeaway: Advances in microsample testing are enabling companies to conduct a whole host of diagnostic testing on a single drop of blood. Advocates say that by minimizing the pain and inconvenience of phlebotomy, more frequent monitoring can provide a better overall picture of patients’ health.