Effect of Blood Collection Tube Type and Time to Processing on the Enumeration and High-Content Characterization of Circulating Tumor Cells Using the High-Definition Single-Cell Assay.
Author(s): Rodríguez-Lee M, Kolatkar A, McCormick M, Dago AD, Kendall J, Carlsson NA, Bethel K, Greenspan EJ, Hwang SE, Waitman KR, Nieva JJ, Hicks J, Kuhn P
Publication: Arch Pathol Lab Med, 2018, Vol. 142, Page 198-207
PubMed ID: 29144792 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of blood collection tube type and processing delay on the enumeration and single-cell characterization of circulating tumor cells (CTCs).
Conclusion of Paper
Specimens in cell-free DNA (cfDNA) tubes had significantly higher numbers of candidate CTCs [high-definition circulating tumor cells (HD-CTCs) and marginal populations] and lower levels of total negative events compared to specimens EDTA, citrate, and heparin tubes. While specimens in cfDNA, EDTA, and heparin tubes had comparable levels of CTC-Small subpopulations, significantly lower levels of CTC-small subpopulations were found in citrated blood. Blood in heparin tubes had the lowest levels of CTC-Ap cells. No differences were found in the total number of negative events detected between specimens processed 24 h and 72 h post-collection, but the number of candidate CTC cells, total cells retained, and all three marginal subpopulations (CTC-Small, CTC-LowCK, and CTC-Ap) was decreased in 72 h post-collection specimens. Eighty-seven of the 93 cells selected for single-cell characterization were successfully amplified and the copy number variation (CNV) profiles of cells isolated from the 24 h CfDNA tubes exhibited the lowest level of variability of the four tube types.
Studies
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Study Purpose
This study investigated the effects of blood collection tube type and processing delay on the enumeration and characterization of circulating tumor cells (CTCs). Blood (40 mL) was collected from 100 patients with either early-stage or metastatic breast cancer in a syringe and distributed into two each of Streck cell-free DNA, EDTA, acid-citrate-dextrose (ACD), and heparin tubes which were shipped to the laboratory at approximately 22°C. One specimen of each tube type was analyzed 24 h post-collection and the other was stored at room temperature and processed 72 h post-collection. White cells were counted using an open vial sampling system analyzer to determine volume of blood required to create slides with a maximum deposition of 3 million white cells per slide. Six to twelve slides were created from each specimen and stored at -80°C until analysis. Slides were immunofluorescently stained with CD45, a cocktail of pan-cytokeratin (CK), CK19, and DAPI antibodies for analysis (details not provided). CK+, CD45- cells with intact DAPI staining that were larger and morphologically distinct from surrounding white cells were classified as high-definition circulating tumor cells (HD-CTCs). Marginal populations included CTCs of the same size or smaller than neighboring WBCs (CTC-Small), CK levels lower than those of HD-CTCs (CTC-LowCK), or cells with a DAPI pattern of nuclear condensation and fragmentation (CTC-Ap). Positive events were defined as the total number of CTC cells (HD-CTC plus marginal populations) detected per total number of retained cells and negative results as the total number of cellular and non-cellular events lacking DAPI nuclear staining per total number of retained cells. Two slides generated from each tube type processed at 24 h were analyzed and cases that had one or more HD-CTCs were selected for collection tube comparisons (n=50). HD-CTCs identified from one patient with late-stage disease were selected for single-cell next-generation sequencing (16-20 cells from each tube type processed at 24 h and from cfDNA tubes processed at 72 h). Cells were captured by micromanipulation and subjected to whole-genome amplification. DNA was measured by spectrophotometry and quality assessed on a bioanalyzer and by agarose gel electrophoresis. Sequencing was performed on an Illumina HiSeq instrument.
Summary of Findings:
Significantly higher numbers of candidate CTCs (HD-CTCs and marginal populations) were detected in blood in cfDNA tubes than EDTA, citrate, or heparin tubes (P<0.001 for all) which demonstrated comparable numbers. Blood in cfDNA tubes had the lowest level of total negative events compared to the other tube types (P<0.001 for all) and subsequently had the highest ratio of positive to negative events detected. The median number of cells retained was not significantly different between blood collected in cfDNA, EDTA, and citrate tubes (4.33, 4.42, and 4.00; respectively), but blood in heparin tubes had 21.4% fewer cells than cfDNA tubes (median 3.40 vs. 4.33, P<0.001). Blood in cfDNA tubes retained higher levels of the CTC-LowCK subpopulation than the other tube types (P<0.001 for all). While blood in cfDNA, EDTA, and heparin tubes had comparable levels of CTC-small subpopulations, lower levels of CTC-small subpopulations were found in blood in citrate tubes (P<0.007 for all). Blood in heparin tubes had the significantly lower levels of CTC-Ap cells compared to the other tube types (P=0.001 for all). No differences were found in the total number of negative events detected between specimens processed 24 h and 72 h post-collection (P=0.61), but the number of candidate CTC cells, total cells retained, and all three marginal subpopulations (CTC-Small, CTC-LowCK, and CTC-Ap) was decreased in 72 h post-collection specimens (P<0.001, P<0.001, P=0.001, P=0.007, P=0.001, and P=0.001; respectively) and subsequently the 24 h specimens had a higher ratio of positive to negative events (P=0.001). Similar results were observed in matched pair analysis of 24 h and 72 h post-collection specimens for numbers of HD-CTCs and the CTC-Small, CTC-LowCK, and CTC-Ap subpopulations (P=0.001, P=0.001, P=0.04, and P=0.009; respectively). For CTC characterization, 93 individual HD-CTCs from one patient with late-stage disease were selected from five evaluated slides. Eighty-seven of the 93 cells were successfully amplified (17 cells from 24 h cfDNA tubes; 17 cells from 24 h EDTA tubes; 17 cells from 24 h citrate tubes; 19 cells from 24 h heparin tubes; and 17 cells from 72 h cfDNA tubes) and a copy number variation (CNV) profile was generated from each specimen except one HD-CTC from a 24 h citrate tube. The CNV profiles of cells isolated from the 24 h cfDNA tubes exhibited the highest level of similarity when compared to the other sample sets (P=0.001 for all).
Biospecimens
Preservative Types
- None (Fresh)
- Streck/BCT
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Cell count/volume Fluorescent microscopy DNA Automated electrophoresis/Bioanalyzer DNA PCR DNA Spectrophotometry DNA Electrophoresis DNA Next generation sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Time at room temperature 24 h
72 h
Biospecimen Acquisition Type of collection container/solution cell-free DNA tube
EDTA tube
acid-citrate-dextrose (ACD) tube
heparin tube