Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

Search BRD Papers

Evaluation of a simple method for storage of blood samples that enables isolation of circulating tumor cells 96 h after sample collection.

Author(s): Apostolou P, Ntanovasilis DA, Papasotiriou I

Publication: J Biol Res (Thessalon), 2017, Vol. 24, Page 11

PubMed ID: 28975085 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of storage duration at 2-8°C on gene and protein expression of CTCs, CD45-negative cells, and spiked-in cancer cells. Blood (40 mL) was collected from two healthy individuals (30-year old male, 28-year old female) and two cancer patients (45-year old female with stage II breast cancer, 54-year old male with stage III colorectal cancer) into a 50 mL centrifuge tube containing 7 mL of 0.02 M EDTA. Tubes were placed on a roller for 30 min, divided into five clean tubes, and then stored at 2–8 °C for 0, 24, 48, 72, or 96 h before processing. A polysucrose solution (4 mL) was added to the tubes and white cells were collected by centrifugation of blood at 2500 x g. White blood cells were incubated for 10 min in lysis buffer to lyse any remaining red blood cells. White cells from healthy individuals were incubated with CD45 magnetic beads at 4°C for 30 min and CD45-negative cells were isolated by magnetic separation. Specimens from cancer patients were incubated with cytokeratin beads (CK4, CK5, CK6, CK8, CK10, CK13, and CK18) at 4°C for 30 min before positive selection of CK-positive cells by magnetic separation. Isolated cells were examined microscopically to determine phenotype and stained with CD45 and pan-cytokeratin antibodies and then DAPI-counterstained for IHC analysis. Total RNA was extracted using an RNeasy Mini Kit. RNA yield was measured spectrophotometrically. Gene expression levels of housekeeping genes (18S rRNA, 28S rRNA, ACTB, and GAPDH), NANOG, POU5F1 (OCT3/4), SOX2, CD34, NES, ERCC1, DHFR, cFOS, cJUN, cMET (HGFR) and EGFR were determined by real-time qRT-PCR. Endpoint PCR was used to determine levels of housekeeping genes (18S rRNA and ACTB), EPCAM, KRT19, PECAM1 (CD31), and CDH2 (N-cadherin). Additional blood specimens from healthy individuals were spiked with ~3,750,000 cells from a colon or breast cancer cell line, divided into five 15 mL tubes containing 1.5 ml of blood and EDTA (~ 750,000 cancer cells/tube), and then stored at 2–8 °C for 0, 24, 48, 72, or 96 h. Specimens were divided into three aliquots (~250,000 cells/0.5 mL blood) and each was analyzed by flow cytometry for EPCAM and CD227 expression.

   Summary of Findings:

   Gene expression in CD45-negative cells did not significantly change during storage at 2-8°C (0-96 h) for one healthy individual (P=0.50) or up to 72 h (P=0.72) for the other individual, but real-time qPCR cycle threshold (Ct) values for non-housekeeping genes were significantly different after 96 h compared with the values at 0 h for the second individual (P<0.05). Gene expression levels in CTCs isolated from cancer patient blood did not differ significantly during the 96 h of storage for either patient (P=0.63 and P=0.76). Endpoint PCR analysis of blood from healthy individuals spiked with EPCAM-positive breast and colon cancer cells expressed EPCAM for all storage times but only expressed KRT19 and CDH2 from 0 to 72 h. Flow cytometry results of blood from healthy individuals revealed no significant differences in EPCAM expression between storage time periods (P=0.50). While changes in side-scatter properties were observed with storage time in specimens spiked with cancer cells, EPCAM expression remained stable (P=0.79 for breast and P=0.5 for colon cancer cells) and cMET was not detected for any of the time points. CD227 expression in breast cancer cells was also unaffected by storage duration. Microscopic analysis revealed no change in the phenotype of the cells after 96 h of storage or in expression of specific markers as determined by IHC.

   Biospecimens

   • Cell - White Cells
   • Bodily Fluid - Blood

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Fluorescent microscopy
   Cell count/volume	Light microscopy
   Cell count/volume	Flow cytometry
   RNA	Real-time qRT-PCR
   RNA	Spectrophotometry
   RNA	RT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	0 h 24 h 48 h 72 h 96 h
   RT-PCR Specific	Targeted nucleic acid	18S rRNA ACTB EPCAM KRT19 PECAM1 (CD31) CDH2 (N-cadherin)
   Real-time qRT-PCR Specific	Targeted nucleic acid	18S rRNA 28S rRNA ACTB GAPDH NANOG POU5F1 (OCT3/4) SOX2 CD34 NES ERCC1 DHFR cFOS cJUN cMET (HGFR) EGFR

   View more