Kinetic River Corp. reported that it has advanced its Arno technology for time-resolved cell analysis, developing technology that enables compensation-free, 12-marker flow cytometry assays with just two lasers and six detectors using time-resolved methods. The technology demonstrated the doubling of the number of markers that is measurable with a fixed set of lasers and detectors. The company said that the technology could additionally support the tripling of number of markers that can be measured with a fixed set of lasers and detectors.
The advancement enables compensation-free flow cytometry, among other applications, and marks the company’s completion of a Phase II Small Business Innovation Research (SBIR) project funded by the National Institutes of Health (NIH).
By discriminating fluorescent markers not just by their color but also by the decay of their fluorescence emissions, or lifetime, the Arno technology avoids the issues of spectral spillover experienced by existing flow cytometers. Kinetic River reported that its approach successfully doubled of the number of markers measurable with a fixed set of lasers and detectors by resolving emissions from fluorescent labels based on time — allowing the “stacking” of multiple sets of labels.
Lifetime-based discrimination allows emissions from multiple markers to be collected by each detector, greatly expanding the analytical power of flow cytometry while minimizing the number of lasers and detectors needed in a system.
The time-resolved cell analysis technology developed at Kinetic River has demonstrated the doubling of the number of markers measurable with a fixed set of lasers and detectors, and it paves the way to a tripling of the same, as illustrated in this diagram. The patented approach is able to do so by resolving emissions from fluorescent labels based on time — allowing the 'stacking' of multiple sets of labels. Courtesy of Kinetic River.
The company showed results from a 12-marker assay on commercial mononuclear cells performed on the Arno using lasers operating at 405 and 488 nm.
“These results cap more than seven years of research and development,” said Giacomo Vacca, president of Kinetic River. “The fact that we were able to perform a 12-marker assay using only two lasers and six detectors is unprecedented. Even more exciting than that is the range of different applications these results open up, from compensation-free flow cytometry to a vastly expanded number of fluorescent labels measurable with, in fact, fewer lasers and detectors than on existing analyzers.”
Flow cytometry is widely used in research and clinical fields spanning tumor biology research, diagnostics, immunophenotyping, and cancer immunotherapy. Fluorescent markers are used to distinguish different cell types, though the spectral emission overlap between the various markers causes many problems — from a limit to the maximum number of markers measurable in any given sample to a high burden on operators and high costs of reagents and controls to perform the compensation procedures typically required using purely spectral methods.
The time-resolved cell analysis technology developed at Kinetic River has applications to compensation-free flow cytometry, highly multiparametric flow cytometry, and flow cytometry with automated autofluorescence removal, the company said.
In the ability of the technology to enable highly multiparameter flow cytometry specifically, Vacca said users and manufacturers alike are poised to benefit. Users will be able to measure more parameters with a single technology; manufacturers will be able to do so with fewer expensive components — namely lasers and detectors.
Results from a 12-marker assay on commercial mononuclear cells performed on the Arno using only two lasers (405 and 488 nm) and six fluorescence detectors. None of the measured parameters needed to be compensated for spectral spillover, and each detector collected emissions from two markers. The detected emissions were then separated by fluorescence lifetime using proprietary algorithms. Boxes and red arrows indicate the gating procedure for isolating cell subpopulations. An enhanced version of this image can be accessed here. Courtesy of Kinetic River.
As the advance is technologically foundational, rather than technique-specific, it has potential benefits in the vast majority of applications for which flow cytometry is used, Vacca said. These include stem cell research, oncology, organ transplantation, microbiology, and drug discovery.
“It’s a very exciting development. Fluorescence lifetime is a physical attribute of the markers and labels that have been used for decades in diagnostic and analytical instruments, yet no one to this point has been able to harness it for use in flow cytometry,” said Art Monk, CEO of Patent Contacts LLC, an adviser to Kinetic River. “Kinetic River’s work in this area has developed novel fluorescence detection technology, proprietary signal analysis algorithms, and precision fluid management systems for leveraging fluorescence lifetime in these important instruments. Further, the company has reduced its research findings to practice in the equipment it has designed and brought to market.”
William Telford of the National Cancer Institute, who collaborated on experiments involved in the work, said, “We performed benchmark measurements on a spectral cytometer using the exact same fluor-antibody conjugates as on the Arno compensation-free analyzer, and the results were very much in agreement. In some cases, the Arno was able to distinguish fluor contributions even where the spectral cytometer struggled due to the near-complete spectral overlap between fluorescent emissions.”
Vacca said that Kinetic River is taking steps toward commercialization, including collaborative efforts to accelerate the transition to market. Less than one year ago, the company announced the successful development of its Colorado automated autofluorescence removal technology. The Colorado features a combination of hardware and algorithm development, and Vacca said that advancements in the programs of both systems were leveraged in the other.
“The Arno and Colorado both benefited from development of the time-resolved technology foundation at Kinetic River. Based on what we have found, we believe that a product that combines, say, highly multiparametric flow cytometry with automated autofluorescence removal is a real possibility,” Vacca said.
The $1.5 million grant was awarded to Kinetic River by the National Institute of General Medical Sciences.