Preservation of Circulating Tumor Cell RNA, Morphology, and Immunoreactivity Using a New Whole Blood Compatible Preservative

摘要

BACKGROUND: Currently, circulating tumor cells (CTCs) can be isolated and enumerated for up to 96 hours following blood draw into a CellSave Preservative Tube. In CellSave, the CTCs are numerically and morphologically preserved, and their immunoreactivity remains sufficiently intact for immunologic enrichment; however RNA is rapidly degraded and lost from CTCs following exposure to CellSave. In general, most fixatives that could be used to collect and preserve blood samples are either incompatible with whole blood, bind up or degrade RNA, or lyse all the cells present. We report here on a new preservative that is compatible with whole blood and preserves CTC morphology and immunoreactivity while leaving the CTC RNA cell associated and intact. This preservative, called CellSecure, enables the enrichment of rare CTCs from blood in a way that facilitates their full immunochemical analysis or extraction of intact CTC RNA for whatever molecular analysis is desired. METHODS: Whole blood was collected in standard EDTA blood collection tubes, spiked with cell lines, and CellSecure was added. In control tubes, no CellSecure was added. Blood was tested either immediately after CellSecure addition or at 24 hour intervals up to 96 hours. Blood was processed using either the microfluidic Harpoon CTC Isolator and CTC Disk (Hrp-Isolator) technology, or using CELL SEARCH?. Cell lines were enriched from blood incubated from 0-96 hours in CellSecure and examined by flow cytometry to quantitate immunofluorescent reactivity of various antigens, immunofluorescently imaged and counted, or had RNA extracted. Extracted RNA was subjected to gel analysis to determine if it was intact or degraded, and probed for quantitative retention of CTC gene expression over time by qPCR. RESULTS: In CellSecure the white blood cell (WBC) antigen CD45 was stable or decreased in immunofluorescence intensity by ~10-40% over 96 hours, depending on the WBC type. Cell lines spiked into whole blood and stored up to 96 hours could be efficiently enriched and imaged from whole blood by either immunologic capture (CELLSEARCH?) or WBC negative depletion using the Hrp-Isolator microfluidic technologies. Morphology and immunoreactivity was preserved over time using CellSecure, although changes in the morphology of CTCs isolated using CELL SEARCH? were observed at 96 hours. Cells that were not preserved (EDTA alone) showed significant RNA degradation over time, and little or no intact RNA could be found when blood was stored in CellSave for 24 hours. In contrast, blood preserved using CellSecure showed intact RNA by gel analysis. qPCR analysis of RNA expressed by CTCs and WBCs showed expression levels at 96 hours that were either unchanged or only minimally different than the levels seen in the control tubes at time zero. CellSecure was found to work optimally with the Hrp-Isolator, up to 72 hours post draw, recovering between 90-100% of spiked cells with sufficient purity to enable direct qPCR analysis of CTC RNA expression levels. DISCUSSION: We have developed a preservative solution that is compatible with whole blood that preserves cells and the cellular antigens we have tested to date to a sufficient level for cell isolation and characterization; and importantly also preserves the RNA expressed by these cells. By keeping the RNA associated with the cell expressing it, we were able to enrich rare cell populations from 7.5 mLs of whole blood with sufficient purity to enable molecular analysis of genes expressed by these rare cell populations. RNA expression levels are stable for at least 96 hours post draw and 90-100% of the spiked rare cell populations could be recovered using the Hrp-Isolator technology for up to 72 hours post draw. This should enable CellSecure to be used in conjunction with the Hrp-Isolator for collection of samples and the subsequent immunological and molecular analysis of CTCs or other rare cell populations on shipped samples.