Abstract
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 we 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.
Highlights
Precise rare-cell technologies require the blood to be processed immediately or be stabilized with fixatives
Once removed from its native environment, a host of degenerative processes including hemolysis, platelet activation, cytokine and oxidative bursts, and neutrophil extracellular trap formation[4] inflict collateral damage to the entire blood specimen. These problems are exacerbated by the extreme rarity and fragility of CTCs5,6 because the target cells are buried in such a hostile environment and due to the breakdown of stringent rare-cell sorting mechanisms when challenged with disintegrated blood cells, extracellular DNA, as well as altered cellular morphology and marker expression[7]
We found that hypothermic storage using the anticoagulant Acid Citrate Dextrose (ACD; Supplementary Fig. 1) had a clear benefit on granulocyte preservation: their viability after 72 h of cold storage (97.5 ± 0.9%, mean ± SD throughout the text unless specified, n = 5; Fig. 1c) was no different from fresh control samples (0 h; 97.5 ± 2.0%, n = 5; Fig. 1c), while room-temperature storage (RT; ~ 22 ° C) resulted in ~ 30% cell death (Fig. 1c)
Summary
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. Once removed from its native environment, a host of degenerative processes including hemolysis, platelet activation, cytokine and oxidative bursts, and neutrophil extracellular trap formation[4] inflict collateral damage to the entire blood specimen These problems are exacerbated by the extreme rarity and fragility of CTCs5,6 because the target cells are buried in such a hostile environment and due to the breakdown of stringent rare-cell sorting mechanisms when challenged with disintegrated blood cells, extracellular DNA, as well as altered cellular morphology and marker expression[7]. Preservation of whole blood in an unaltered state is critical for acquiring clinically actionable information such as gene expression profiling as well as establishing ex vivo cultures and xenograft models[3]
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