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Blood samples
Photo by Graham Colm

A new blood stabilization method significantly prolongs the lifespan of blood samples for microfluidic sorting and transcriptome profiling of rare circulating tumor cells (CTCs), according to researchers.

The method involves reducing the storage temperature to 4° C to reversibly slow down cellular processes while also counteracting the platelet activation that can occur because of the low temperature.

The researchers said this work overcomes a significant barrier to the translation of liquid biopsy technologies for precision oncology and other applications.

Keith Wong, PhD, of the Massachusetts General Hospital Center for Engineering in Medicine (MGH-CEM), and his colleagues described this work in Nature Communications.

When isolating CTCs from fresh, unprocessed blood, timing is everything. Even minor changes in the quality of a blood sample—such as the breakdown of red cells, leukocyte activation, or clot formation— can greatly affect cell-sorting mechanisms and the quality of the biomolecules isolated for cancer detection.

According to published studies, factors such as the total number of CTCs in a sample and the number with high-quality RNA decrease by around 50% within the first 4 to 5 hours after the sample is collected.

“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 2 after they are drawn,” Dr Wong said.

“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.”

With this in mind, Dr Wong and his colleagues set out to preserve blood in its native state with minimal alterations.

“We wanted to slow down the biological clock as much as possible by using hypothermia, but that is not as simple as it sounds,” said study author Shannon Tessier, PhD, also of MGH-CEM.

“Low temperature is a powerful means to decrease metabolism, but a host of unwanted side effects occur at the same time. In some ways, these challenges are similar to those we face in organ preservation, where we have to optimize strategies for a very complex mix of cells.”

To achieve these goals, the researchers first analyzed the effects of hypothermic storage conditions.

The team found that hypothermic storage (4° C) of blood anticoagulated with acid citrate dextrose maintained “cellular morphology, integrity, and surface epitope stability of diverse hematologic cell types” over 72 hours.

However, the researchers also observed platelet activation.

“We are preserving the blood very well, including the coagulation function of platelets,” Dr Wong said. “But, unfortunately, cooling causes profound activation of platelets. Now, we need a targeted approach for platelets so they don’t form nasty clots in the microfluidic blood-sorting device.”

Fortunately, the researchers found that glycoprotein IIb/IIIa inhibitors were able to counter cooling-induced platelet aggregation. And ion chelation treatment with ethylenediaminetetraacetic acid removed activated platelets from leukocytes.

The team said these steps—hypothermic storage, treatment with glycoprotein IIb/IIIa inhibitors, and ion chelation—allowed whole blood preserved for 3 days to be processed as if it were freshly drawn, with very high purity and virtually no loss in CTC numbers.

“The critical achievement here 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,” Dr Tessier said.

To test their blood preservation method, Dr Tessier and her colleagues used blood specimens from a group of 10 patients with metastatic prostate cancer.

The researchers compared CTC analysis in preserved blood samples and paired fresh samples from the same patients.

There was 92% agreement in the detection of 12 cancer-specific gene transcripts between the fresh and preserved blood samples.

In addition, there was 100% agreement in the detection of a transcript called AR-V7. Recently published studies showed that the presence of AR-V7 mRNA in prostate cancer CTCs predicts resistance to androgen receptor inhibitors, indicating that chemotherapy may be a better option for such patients.

“The ability to preserve the blood for several days and still be able to pick up this clinically relevant biomarker is remarkable,” said study author David Miyamoto, MD, PhD, of MGH Cancer Center.

“This is very exciting for clinicians because AR-V7 mRNA can only be detected using CTCs and not with circulating tumor DNA or other cell-free assays.”

The researchers highlighted the universal nature of their blood preservation approach by pointing to its compatibility with the microfluidic CTC-iChip device, which isolates tumor cells by rapid removal of blood cells. The team said this suggests the potential impact of this work extends beyond cancer detection.

“With exciting breakthroughs in immunotherapy, stem cell transplantation, and regenerative medicine—in which peripheral blood is often the source of cells for functional assays or ex vivo expansion—the ability to preserve live cells will greatly ease logistical timelines and reduce the cost of complex cell-based assays,” Dr Wong said.


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