A new work published in Nature Communications reported a new blood stabilization method significantly prolongs the lifespan of blood samples for microfluidic sorting and transcriptome profiling of rare circulating tumour cells (CTCs) carried in the bloodstream.

The only FDA-approved blood stabilization method for CTC assays is a chemical fixation, which kills the cells and heavily degrades sensitive biomolecules, especially RNA. Chemically fixing the cells defeats the purpose of using them in clinically meaningful ways. When isolating these extremely fragile and rare cells from fresh, unprocessed blood, timing is everything.

Even minor changes in the quality of a blood sample greatly affect cell-sorting mechanisms and the quality of the biomolecules isolated for cancer detection. Important 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 four to five hours after the sample is taken.

The team took a comprehensive approach that aims to preserve blood in its native state with minimal alterations. The researchers wanted to slow down the biological clock as much as possible by using hypothermia. Low temperature is a powerful means to decrease metabolism, but a host of unwanted side effects occur at the same time.

The team analyzed the storage conditions that optimally preserve the viability of the diverse cell types in whole blood. The biggest challenge was platelet activation. The team preserved the blood very well, including the coagulation function of platelets. But, cooling causes profound activation of platelets. A targeted approach required for platelets so they don't form nasty clots in the microfluidic blood sorting device.

The team then analyzed a variety of antiplatelet agents and found that glycoprotein IIb/IIIa inhibitors were extremely effective in countering cooling-induced platelet aggregation. These strategies allow whole blood preserved for three days to be processed as if it were freshly drawn, with very high purity and virtually no loss in the number of CTCs.

The critical achievement is that the isolated tumour cells contain high-quality RNA that is suitable for demanding molecular assays, such as single-cell qPCR, droplet digital PCR and RNA sequencing. Using blood specimens from a group of 10 patients with metastatic prostate cancer, the team compared the use of preserved blood against paired fresh samples from the same patients for CTC analysis.

There was 92% agreement in the detection of 12 cancer-specific gene transcripts between the fresh and the preserved samples, and there was 100% agreement in the detection of a transcript called AR-V7 mRNA. The mRNA can only be detected using CTCs and not with circulating tumour DNA or other cell-free assays.

The team highlights the universal nature of the stabilization approach by pointing to its compatibility with the highly demanding microfluidic CTC-iChip device, which isolates tumour cells by rapid removal of blood cells, implying the potential impact of the work extends beyond cancer detection.