Using GPIIb/IIIa inhibition, and ion chelation, researchers have developed a “universal” method for preserving blood up to 72 hours while keeping it viable for advanced rare-cell applications
Through microfluidics and automation, clinical laboratories and anatomic pathologists have been able to detect ever-smaller quantities of biomarkers and other indicators of chronic disease.
However, preserving sample quality is an essential part of analytical accuracy. This is particularly true in precision oncology and other specialties where isolating rare cells (aka, low abundance cells), such as circulating tumor cells (CTCs), is a key component to obtaining information and running diagnostics.
Publishing their finding in Nature, researchers at Massachusetts General Hospital Center for Engineering in Medicine (MGH-CEM) have developed a whole blood stabilization method that is ideal for rare-cell applications, and which preserves sample integrity for up to 72 hours.
Should further testing validate their findings and methodology, this change could allow greater use of central laboratories and other remote testing facilities that previously would not be available due to distance and sample travel time.
Keeping Blood Alive Is Not Easy
“At Mass. General, we have the luxury of being so integrated with the clinical team that we can process blood specimens in the lab typically within an hour or two after they are drawn,” stated lead author Keith Wong, PhD, former Research Fellow, MGH-CEM, and now Senior Scientist at Rubius Therapeutics, Boston, in a Mass General press release. “But to make these liquid biopsy technologies routine lab tests for the rest of the world, we need ways to keep blood alive for much longer than several hours, since these assays are best performed in central laboratories for reasons of cost-effectiveness and reproducibility.”
Study authors Wong and co-lead author Shannon Tessier, PhD, Investigator at MGH-CEM, noted that current FDA-approved blood stabilization methods for CTC assays use chemical fixation—a process that can result in degradation of sensitive biomolecules and kill the cells within the sample.
Without stabilization, however, breakdown of red cells, activation of leukocytes (white blood cells), and clot formation can render the results of analyzing a sample useless, or create issues with increasingly sensitive equipment used to run assays and diagnostics.
“We wanted to slow down the biological clock as much as possible by using hypothermia, but that is not as simple as it sounds,” says Tessier. “Low temperature is a powerful means to decrease metabolism, but a host of unwanted side effects occur at the same time.”
Researchers started by using hypothermic treatments to slow degradation and cell death. However, this created another obstacle—aggressive platelet coagulation. By introducing glycoprotein IIb/IIIa inhibitors, they found they could minimize this aggregation.
Prior to microfluidic processing of their test samples, researchers applied a brief calcium chelation treatment. The result was efficient sorting of rare CTCs from blood drawn up to 72 hours prior, while keeping RNA intact and retaining cell viability.
“The critical achievement here,” says Tessier, “Is that the isolated tumor cells contain high-quality RNA that is suitable for demanding molecular assays, such as single-cell qPCR, droplet digital PCR, and RNA sequencing.”
Their testing involved 10 patients with metastatic prostate cancer. Sample integrity was verified by comparing CTC analysis results between fresh samples and preserved samples from the same patients using MGH-CEM’s own microfluidic CTC-iChip device.
Results showed a 92% agreement across 12 cancer-specific gene transcripts. For AR-V7, their preservation method achieved 100% agreement. “This is very exciting for clinicians,” declared David Miyamoto, MD, PhD, of Massachusetts General Hospital Cancer Center in the press release. “AR-V7 mRNA can only be detected using CTCs and not with circulating tumor DNA or other cell-free assays.”
Methodology Concerns and Future Confirmations
“Moving forward, an extremely exciting area in precision oncology is the establishment of patient-specific CTC cultures and xenograft models for drug susceptibility,” the study authors noted. “The lack of robust methods to preserve viable CTCs is a major roadblock towards this Holy Grail in liquid biopsy. In our preliminary experiments, we found that spiked tumor cells in blood remain highly viable (>80%) after 72 hours of hypothermic preservation.”
Despite this, they also acknowledge limitations on their current findings. The first is the need for larger-scale validation, as their testing involved a 10-patient sample group.
Second, they note that further studies will be needed to “more completely characterize whole-transcriptome alterations as a result of preservation, and to what extent they can be stabilized through other means, such as further cooling (e.g., non-freezing sub-zero temperatures) or metabolic depression.”
Researchers also note that their approach has multiple advantages for regulatory approval and further testing—GPIIb/IIIa inhibitors are both low-cost and already approved for clinical use, implementation requires no modification of existing isolation assays, and cold chain protocols are already in place allowing for easy adaptation to fit the needs of pathology groups, medical laboratories, and other diagnostics providers handling samples.
While still in its early stages, the methods introduced by the researchers at MGH-CEM show potential to allow both the facilities collecting samples and the clinical laboratories processing them greater flexibility and increased accuracy, as high-sensitivity assays and diagnostics continue to power the push toward personalized medicine and expand laboratory menus across the industry.
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