The effect of storage on the survival of cancer cells in blood and efficient elimination of contaminating cancer cells by a leukocyte depletion filter.
Author(s): Futamura N, Nakanishi H, Hirose H, Nakamura S, Tatematsu M
Publication: Am Surg, 2005, Vol. 71, Page 585-90
PubMed ID: 16089124 PubMed Review Paper? No
Purpose of Paper
The paper assessed the detectability of tumor cells in whole blood immediately and after storage for 21 days at 4°C by RT-PCR amplification of cytokeratin 19 (CK-19). The efficiency of nucleated cell removal from blood specimens by leukocyte filtration was also investigated.
Conclusion of Paper
CK-19 mRNA was detected by RT-PCR in blood spiked with as few as 102 cancer cells in 2 mL of blood. CK-19 mRNA remained detectable after blood storage for up to 21 days at 4°C, although the number of cells decreased after 14 days of storage. Leukocyte depletion by filtration was highly effective in the removal of cancer cells from blood as determined by the absence of a CK-19 RT-PCR amplicon in spiked specimens.
Studies
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Study Purpose
This study assessed the detectability of CK-19 positive cancer cells in whole blood immediately and after storage at 4°C for up to 21 days, as well as examined the efficiency of cancer cell removal from blood by leukocyte filtration. Blood was collected from healthy volunteers and ACD-A solution (0.15 mL to 1 mL of blood) was added as a preservative. To determine if prolonged storage of whole blood affects cancer cell survival and detectability, cultured cancer cells (at a concentration of 105) from a differentiated gastric carcinoma cell line (MKN-74) or a moderately differentiated colonic carcinoma cell line (COLM-2) were added to 2 mL of blood and stored at 4°C. Red blood cells were lysed and RNA was extracted from the nucleated cell fraction before, immediately after, and on 1, 7, 14, and 21 days after addition of cancer cells. CK-19 mRNA was examined by a nested RT-PCR method followed by Southern blot hybridization using GAPDH as a housekeeping gene. To determine sensitivity, 10-fold serial dilutions of cancer cell lines were mixed with 2 mL of blood and total RNA was isolated and analyzed for CK-19 mRNA. To examine the effectiveness of leukocyte filtration on removal of cancer cells, blood specimens (2 mL) mixed with Ethachinmate (a carrier used to improve recovery of RNA after filtration) were screened for a CK-19 RT-PCR amplicon before and after filtration through an Immuguard III-RC filter.
Summary of Findings:
CK-19 mRNA was detected in blood spiked with cancer cells at concentrations of 105 to 102 in 2 mL of blood for both COLM-2 and MKN-74 cancer cell lines. CK-19 mRNA was also detected in blood stored for up to 21 days at 4°C, although semiquantification of cancer cell survival determined that the number of cells decreased from 105 to 104 after storage at 4°C for 14 days and to 102 – 103 in blood stored for 21 days. Neither CK-19 nor GAPDH mRNA was detected in blood specimens spiked with cells from either cancer line (COLM-2 or MKN-74) after filtration, suggesting the method was effective in removing leukocytes from blood.
Biospecimens
Preservative Types
- Other Preservative
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Southern blot RNA RT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Cell number number of cancer cells/2mL blood
Biospecimen Aliquots and Components Filtration leukocyte depletion filter
unfiltered
Southern blot Specific Targeted nucleic acid CK-19
GAPDH
RT-PCR Specific Targeted nucleic acid CK-19
GAPDH
Storage Storage duration 0 days
1 day
7 days
14 days
21 days