Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Cryopreservation of Circulating Tumor Cells for Enumeration and Characterization.

Author(s): Nejlund S, Smith J, Kraan J, Stender H, Van MN, Langkjer ST, Nielsen MT, Sölétormos G, Hillig T

Publication: Biopreserv Biobank, 2016, Vol. 14, Page 330-7

PubMed ID: 27092845 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of blood collection tube type, frozen storage of buffy coat, and frozen storage duration on enumeration and characterization of tumor cells spiked into blood from healthy volunteers. Intra-patient variation and effects of frozen storage on enumeration of CTCs in blood from breast cancer patients were also examined. Peripheral blood (7.5 mL) was collected from healthy volunteers in EDTA or CellSave tubes and spiked with ~50 or ~500 cells from breast cancer cell lines (MCF-7, SK-BR-3, and MDA-MB-231). Specimens were centrifuged at 2500 x g at 4°C for 15 minutes and buffy coat layers were collected for immediate staining (anti-CD45, DAPI nuclear stain, anti-CK, and anti-EpCAM) and characterization by flow cytometry or enumeration by CytoTrack (ultra-sensitive scanning fluorescence microscopy) or stored at -80°C for 1 day to up to 6 months before analysis. Frozen specimens were thawed in a water bath at 37°C for ~3 minutes before staining and analysis. Criteria to be considered a CTC was CD45-negative, round cells >4 mm with a DAPI-stained nucleus associated with at least 50% CK-positive cytoplasm. To examine inter-patient variation, paired blood specimens were collected in CellSave tubes from 64 patients with invasive breast cancer, stored at room temperature, and analyzed for CTC enumeration within 96 hours. To examine the effects of frozen storage duration on CTC recovery, 17 paired specimens were collected in CellSave tubes from breast cancer patients, processed as described above for spiked-in cells, and analyzed within 96 hours or stored at -80°C for 3-90 days before analysis.

   Summary of Findings:

   Cytokeratin, EpCam, and CD45 expression on recovered spiked-in cells were not affected by frozen storage, regardless of blood collection tube type. CytoTrack analysis of EDTA and CellSave specimens spiked with ~500 cells and stored frozen for one day demonstrated a 90% cell recovery rate compared to fresh specimens (455 vs. 501 cells and 429 vs. 482 cells, respectively). Specimens spiked with 21-53 cells (median 43) demonstrated a 63% recovery rate during 6 months of frozen storage (median recovery of 27 cells, range 13–34 cells, P=0.03) while cell recovery rates from specimens spiked with 0-14 cells (median 3) were equivalent to fresh specimens (median recovery 8 cells, range 0–22, P=0.09). For breast cancer patients, intra-individual variation in paired, fresh specimens was high (r= 0.59, P<0.0001) and the number of CTCs detected was either increased or decreased after frozen storage of up to 90 days compared to the paired fresh specimen, but the differences were not significant.

   Biospecimens

   • Bodily Fluid - Buffy Coat
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen
   • None (Fresh)

   Diagnoses:

   • Neoplastic - Carcinoma
   • Normal

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Fluorescent microscopy
   Protein	Flow cytometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Type of collection container/solution	EDTA blood tube CellSave tube
   Biospecimen Aliquots and Components	Cell number	~50 spiked-in cells ~500 spiked-in cells
   Flow cytometry Specific	Targeted peptide/protein	Cytokeratin CD45 EpCAM
   Storage	Storage duration	0 days 1 day-6 months 3-90 days
   Biospecimen Preservation	Type of fixation/preservation	None (fresh) Frozen

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