NEW YORK (GenomeWeb) – With the goal of developing a novel molecular diagnostic assay to aid in the lung transplantation process, researchers from the University of Toronto have published a new study describing the development of a chip for assessing biomarkers of lung tissue health using a technology called fractal circuit sensors (FraCS).
In their study, published late last week in Science Advances, the team led by U Toronto's Shana Kelley showed that a proof-of-concept lung tissue assessment chip enabled by these FraCS was able to rapidly assess predictive mRNA biomarkers in donor lung tissue samples obtained by surgeons as part of the transplantation process.
Lung transplantation patients are at risk of a complication called primary graft dysfunction, which causes severe injury to the transplanted lung and is responsible for approximately one-third of deaths following the procedure. Physicians currently use various clinical tools to assess which donor lungs are likely to cause PGD, but the field lacks a truly accurate means for such an assessment.
Though microarrays and PCR have been powerful tools for the discovery and validation of molecular biomarkers in the context of lung and other organ transplants, these technologies — requiring numerous sample purification steps and lengthy turnaround times — aren't suitable for the clinical setting in which a lung transplant assay would be used — an approximately two-hour window after a donor organ initially becomes available.
In recognition of this shortfall, Kelley told GenomeWeb this week that she and colleagues hoped to develop a nanoscale sensor technology that could enable rapid and simple electrochemical detection of biomarkers linked to PGD.
Kelley and her team have been involved in the development of a number of different electrochemical sensor technologies in the MDx space, including a recent project using clamp molecules for the detection of mutations in circulating cell-free DNA, as well as systems for the detection of infectious diseases, which they have previously licensed to MDx company Xagenic.
However, the types of technologies that enable rapid, point-of-care infectious disease detection don't have the sensitivity and quantitative ability to distinguish the small differences in analyte levels that are necessary in the context of mRNA-based transplant assessment, she said.
Working with colleagues from the Toronto University Health Network, Kelley and her team set out to develop a lung transplant biomarker chip that would be capable of reporting donor lung biomarker profiles in about 20 minutes using a class of electrochemical biosensors they designed to rapidly capture and analyze non-amplified mRNA transcripts.
The team's FraCS technology comprises a glass microchip that contains electrodeposited gold sensors. According to the authors, the chip is developed using electrodeposition in a way that facilitates rapid growth and the generation of "spiky, fractal structures extending into solution."
"Here we're really trying to quantify the levels of mRNAs that our collaborators identified as being predictive of how a lung will do in a recipient post-transplant," Kelley explained. "Our previous sensors offer an on/off answer — is an analyte present or absent? But here we have to be able to tell whether a transcript is present at a level that you would expect for healthy tissue or is elevated, indicating that there is something bad going on with the tissue."
"To do that we made the sensors really big," she said. "We generated these templates that are basically lines that we could deposit a sensor into, so we have these really large surface areas."
In their study, Kelley and her colleagues first developed probes against three mRNAs previously reported as donor lung assessment markers (IL-6, IL-10, and ATP11B) as well as control sequences to create a prototype lung transplant assessment FraCS-based chip, and set out to compare it with the current gold standard for quantitative gene expression profiling — quantitative PCR.
The group tested 23 donor lungs using both the FraCS method and a Thermo Fisher Scientific Applied Biosystems qPCR platform, and compared the resulting expression patterns. For each of the biomarkers tested (IL-6, IL-10, and ATP11B), the authors wrote that they observed the same expression pattern in both FraCS and qPCR, indicating that the FraCS chip-based approach yields comparable data to qPCR but without the associated sample prep and time requirements.
Finally the team applied its FraCS chip approach to a set of donor lung samples, to evaluate its ability to distinguish lung tissue associated with different ultimate transplant outcomes, and to validate a set of putative transplant suitability markers.
Measuring the expression profiles of IL-6, IL-10, IL-6/IL-10 ratio, and ATP11B, the researchers found that there was a significant difference between the lungs that were later associated with mild or no PGD symptoms versus those that were implicated in severe PGD. This was true for IL-6, IL-6/IL-10, and ATP11B, but not for IL-10.
According to the authors, this finding was consistent with previous work that also determined that IL-10 alone was not a predictor of poor outcome but that as part of the IL-6/IL-10 ratio, the metric is indeed predictive of PGD.
Using gene expression profiles from 32 donor lungs, the investigators then performed statistical analysis using an algorithm they developed called the FraCS prediction model, incorporating the output for the IL-6/IL-10 ratio, IL-6, and ATP11B, which was the most predictive of later PGD development. When they validated this model on 20 new, previously untested samples, the team saw an AUC of 0.87.
Kelley said she and her coauthors now plan to move on to a much larger validation study with their collaborators at the Toronto General Hospital, which has a large repository of samples.
"We are in good shape … to be able to crank through a lot of samples to make sure our algorithm is good enough to be used," she said.
In addition, the team is planning to begin working on some prototype instruments, "to figure out what the essential components are to take a biopsy sample and get an answer out in 20 minutes," Kelley added.
In their initial study the researchers were looking primarily at the FraCS method's ability to distinguish transplanted lungs that went on to do well versus those that did not.
But the technology also has the potential to be able to provide information to help identify viable lungs that may be discarded before ever being transplanted based on current clinical evaluation strategies.
"Only about 15 percent of the lungs that get donated currently actually get used," Kelley said. "There is a big pool of lungs that fail other assessments and using our approach we may be able to give clinicians some other information," potentially expanding the pool of usable organs.
According to the investigators, more than 40 percent of discarded donor lungs may in fact be suitable for safe and successful transplantation.