Whole blood stabilization for the microfluidic isolation and molecular characterization of circulating tumor cells

Precise rare-cell technologies require the blood to be processed immediately or be stabilized with fixatives. Such restrictions limit the translation of circulating tumor cell (CTC)-based liquid biopsy assays that provide accurate molecular data in guiding clinical decisions. Here researchers from Harvard Medical School describe a method to preserve whole blood in its minimally altered state by combining hypothermic preservation with targeted strategies that counter cooling-induced platelet activation. Using this method, whole blood preserved for up to 72 h can be readily processed for microfluidic sorting without compromising CTC yield and viability. The tumor cells retain high-quality intact RNA suitable for single-cell RT-qPCR as well as RNA-Seq, enabling the reliable detection of cancer-specific transcripts including the androgen-receptor splice variant 7 in a cohort of prostate cancer patients with an overall concordance of 92% between fresh and preserved blood. This work will serve as a springboard for the dissemination of diverse blood-based diagnostics.

Functional operation of microfluidic CTC isolation requires stabilized whole blood


a Representative images of the micropost array that performs size-based sorting (debulking) in the CTC-iChip. Blood storage in room temperature, even if treated with tiro-EDTA, results in aggregates that contain sheared DNA consistent with cell death and extracellular trap formation. Cold storage without tiro-EDTA leads to clots that contain densely packed platelets (CD61 staining) and intact cells. In both cases, rare cells are trapped within the aggregates. Cold storage with tiro-EDTA consistently permits clean processing (inset). b CTC isolation performance of the CTC-iChip in different storage conditions. All scale bars represent 50 μm. Box-and-whiskers plots show median, interquartile range, maxima, and minima. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (one-way ANOVA followed by Tukey’s post test)

Wong KHK, Tessier SN, Miyamoto DT, Miller KL, Bookstaver LD, Carey TR, Stannard CJ, Thapar V, Tai EC, Vo KD, Emmons ES, Pleskow HM, Sandlin RD, Sequist LV, Ting DT, Haber DA, Maheswaran S, Stott SL, Toner M. (2017) Whole blood stabilization for the microfluidic isolation and molecular characterization of circulating tumor cells. Nat Commun 8(1):1733. [article]

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