Assessment of pre-analytical sample handling conditions for comprehensive liquid biopsy analyses.
Author(s): Gerber T, Taschner-Mandl S, Saloberger-Sindhöringer L, Popitsch N, Heitzer E, Witt V, Geyeregger R, Hutter C, Schwentner R, Ambros IM, Ambros PF
Publication: J Mol Diagn, 2020, Vol. , Page
PubMed ID: 32497717 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to investigate the effect of tube type, pre- and post-centrifugation storage, and blood processing method on the cell free DNA (cfDNA) yield, size distribution, next-generation sequencing (NGS) metrics, and cell profile.
Conclusion of Paper
Although cfDNA levels were comparable in plasma from blood collected in PAXgene ccfDNA, Streck BCT, Roche cfDNA, and EDTA tubes, cfDNA levels were higher and the ratio of MYCN/NAGK more variable in heparin plasma. Serum had higher levels of cfDNA and lower MYCN/NAGK ratios than PAXgene or EDTA plasma. cfDNA yield was stable when blood was stored at room temperature for up to 48 h in PAXgene ccfDNA, Streck BCT, or Roche cfDNA tubes or when stored at 4°C in EDTA tubes or clot activator tubes but cfDNA yield increased with room temperature storage of blood in heparin, EDTA tubes, or clot activator tubes. Storage of serum isolated from clot activator tubes resulted in a small but significant increase in cfDNA but storage of serum from gel tubes resulted in increasing variability in DNA yield and a significant increase in DNA contamination after 72 h at room temperature.
Library yield, percentage mapped reads, and duplication rates were comparable in plasma and serum specimens and when plasma was obtained by centrifugation and density gradient centrifugation. The calculated tumor fraction was lower in the serum specimen than the matched plasma specimen.
Cell profiles were dependent on tube type, processing delay, and processing method but was less affected by tube type and processing delay when plasma was obtained by density gradient centrifugation.
Studies
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Study Purpose
The purpose of the study was to compare cfDNA yield, size distribution, and ddPCR metrics in plasma from blood collected and stored in different tube types. Blood was collected from 22 healthy volunteers by venipuncture into Streck cell-free DNA BCT CE tubes, Roche cell-free DNA collection tubes, Lithium heparin tubes, KEDTA S-MONOVETTE tubes, and PAXgene Blood ccfDNA tubes and stored for 2, 4, 12, 24, or 48 hours at room temperature or in EDTA tubes at 4°C before centrifugation. After addition of buffered formaldehyde to the K2EDTA tubes, plasma was obtained by centrifugation at 1600 x g for 10 min at 4°C twice and then stored at -80°C until DNA extraction. cfDNA was isolated from plasma using the QIAamp Circulating Nucleic Acid Kit, quantified using Qubit dsDNA Assay Kit, and the size distribution was evaluated using the High Sensitivity DNA 5000 Kit on a TapeStation. MYCN and NAGK copy number were further quantified by droplet digital PCR (ddPCR) and real-time PCR.
Summary of Findings:
Although cfDNA levels were comparable in plasma from blood collected in PAXgene ccfDNA, Streck BCT, Roche cfDNA, or EDTA tubes; they were 2.4-fold higher when blood was collected in heparin tubes. Further, ddPCR failed in 5 of 15 specimens and the ratio of MYCN/NAGK was more variable when collected in heparin tubes compared to all other tube types. cfDNA yield was stable when blood was stored at room temperature for up to 48 h in PAXgene ccfDNA, Streck BCT, or Roche cfDNA tubes but there was a significant increase in cfDNA yield after 24 h in heparin tubes (17-fold, P<0.0001) or EDTA tubes (approximately 2-fold, P<0.0001). Along with the increase in cfDNA yield in heparin and EDTA tubes, there was a decrease in the abundance of short fragments at 24 and 48 h, respectively. When EDTA blood was stored at 4°C instead of room temperature, levels of cfDNA and the relative abundance of short fragments remained stable for the 24 h period evaluated. In addition, storage of blood for 24 h at room temperature in Streck BCT or EDTA tubes or for 48 h in Roche cfDNA tubes resulted in increased variability in the ratio of MYCN/NAGK but the number of informative droplets remained stable.
Biospecimens
Preservative Types
- Other Preservative
- None (Fresh)
- Streck/BCT
- PAXgene
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Automated electrophoresis/Bioanalyzer DNA Digital PCR DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution Streck cell-free DNA BCT CE
Roche cell-free DNA collection tube
Lithium heparin
KEDTA S-MONOVETTE tubes
PAXgene Blood ccfDNA tubes
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Storage Storage duration 4 h
12 h
24 h
48 h
0 h
2 h
Storage Storage temperature Room temperature
4°C
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Study Purpose
The purpose of this study was to investigate the effects of tube type and pre-and post-centrifugation storage on the yield and size distribution of cfDNA from serum. Blood was collected from 22 healthy volunteers by venipuncture into S-Monovette tubes with clot activator and S-Monovette Gel-Serum tubes containing polyacrylic ester gel and allowed to clot for 30 min before storage for 0, 3, 12, 24 48 and 72 h at room temperature or for 0, 12, 24, 48 and 72 hat 4°C. Serum was obtained by centrifugation at 2000 x g for 10 min at room temperature and transferred to a new tube and frozen at -80°C. Some specimens clotted at room temperature for 30 min and were stored 3, 12, or 24 h before freezing. cfDNA was isolated from serum using the QIAamp Circulating Nucleic Acid Kit. cfDNA was quantified using Qubit dsDNA Assay Kit and the size distribution was evaluated using the High Sensitivity DNA 5000 Kit on a TapeStation.
Summary of Findings:
Pre-centrifugation storage of blood at room temperature in clot activator tubes for ≥3 h (P<0.05) but not at 4°C resulted in an increase in DNA yield and an increase in small fragments. Storage of serum isolated from clot activator tubes for ≥12 h at either temperature resulted in a small but significant increase in cfDNA (P<0.05). In contrast, while storage of serum from gel tubes resulted in increasing variability in DNA yield, a significant increase in DNA contamination was only observed after 72 h at room temperature (P<0.05) and size distribution remained unaffected.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Automated electrophoresis/Bioanalyzer DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Biospecimen Acquisition Type of collection container/solution S-Monovette tubes with clot activator
S-Monovette Gel-Serum tubes containing polyacrylic ester gel
Storage Storage conditions As blood
As serum
Storage Storage duration 0 h
3 h
12 h
24 h
48 h
72 h
Storage Storage temperature Room temperature
4°C
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Study Purpose
The purpose of this study was to compare recovery of DNA from plasma and serum obtained from blood collected and stored in different tubes type. Blood was collected from three healthy volunteers by venipuncture into S-Monovette tubes with clot activator, PAXgene ccfDNA tubes, and EDTA tubes. Specimens were spiked with 10 ng/mL fragmented DNA from a neuroblastoma cell line and stored for 4, 24, or 48 h at 4°C. Serum was obtained by centrifugation at 2000 x g for 10 min at room temperature and plasma by centrifugation at 1600 x g for 10 min at 4°C twice. cfDNA was extracted using the QIAamp Circulating Nucleic Acid Kit. cfDNA was quantified using the Qubit dsDNA Assay Kit and the size distribution was evaluated using the High Sensitivity DNA 5000 Kit on a TapeStation. MYCN and NAGK copy number were further quantified by droplet digital PCR and real-time PCR MYCN and NAGK.
Summary of Findings:
Although levels of cfDNA were comparable and stable in EDTA and PAXgene specimens stored for up to 48 h before centrifugation, levels were higher in serum specimens than PAXgene tubes stored for 0, 24, or 48 h (P<0.05, P<0.01, and P<0.001; respectively) and EDTA tubes stored for 24 or 48 h (P<0.05 and P<0.01, respectively). Further, the MYCN/NAGK ratio was significantly lower in serum specimens then EDTA and PAXgene specimens and fragment analysis showed a shift toward longer fragments.
Biospecimens
Preservative Types
- None (Fresh)
- Other Preservative
- PAXgene
- Streck/BCT
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Fluorometry DNA Digital PCR DNA Automated electrophoresis/Bioanalyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution S-Monovette tubes with clot activator
PAXgene ccfDNA
EDTA tubes
Storage Storage duration 4 h
24 h
48 h
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
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Study Purpose
The purpose of this study was to compare NGS sequencing metrics and tumor fraction in serum and plasma specimens. Blood specimens from three patients with neuroblastoma were collected and stored in EDTA tubes for approximately 4 h at room temperature and serum tubes for 24 h at room temperature before processing. Serum was obtained by centrifugation at 2000 x g for 10 min at room temperature and plasma by centrifugation at 1600 x g for 10 min at 4°C twice. Plasma and serum were stored at -80°C before cfDNA extraction. cfDNA was isolated from serum and plasma using the QIAamp Circulating Nucleic Acid Kit. Sequencing libraries were prepared with the NEBNext UltraTM II DNA Library Prep Kit for Illumina and sequenced on an Illumina HiSeq 3000 with single-end and 50bp or 65bp reads.
Summary of Findings:
Library yield, percentage mapped reads, and duplication rates were comparable in plasma and serum specimens but the calculated tumor fraction for all three patients was lower in the serum specimen than the matched plasma specimens (0.57 versus 0.31, 0.79 versus 0.38, and 0.54 versus 0.08) and only a flat profile was visible for the serum specimen for one patient.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Neoplastic - Sarcoma
Platform:
Analyte Technology Platform DNA Next generation sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Blood and blood products Plasma
Serum
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Study Purpose
The purpose of this study was to compare cell counts in PBMC obtained by density gradient centrifugation and by centrifugation of blood collected and stored in EDTA, Roche cfDNA, PAXgene ccfDNA, and Streck BCT tubes. Blood was collected from three healthy volunteers by venipuncture into EDTA, Roche cfDNA, PAXgene ccfDNA, and Streck BCT tubes and processed after 0 or 24 h at 4°C. Half of each specimen was centrifuged twice at 1600 x g for 10 min at 4°C to obtain plasma. PBMCs were isolated from the other half of the specimen using density gradient centrifugation using Lymphoprep. Blood cell counts were assessed using a Sysmex analyzer and further evaluated by flow cytometry after staining with antibodies for CD45, CD7, CD19, CD3, CD4, CD8, CD14, CD16, CD19, CD56, αβTCR, and γδTCR.
Summary of Findings:
Whole blood cell counts were comparable between tubes types when processed immediately and when subjected to a 24 h delay in centrifugation. When subjected to density gradient centrifugation, cells stored in EDTA tubes for 24 h had a higher number of lymphocytes than the other specimen types (P<0.001, all) and specimens in PAXgene tubes retained granulocytes. When obtained by centrifugation, the number of lymphocytes and granulocytes were higher in Roche cfDNA and EDTA specimens than in to PAXgene ccfDNA and Streck BCT when processed without delay but not when stored for 24 h. When processed immediately by centrifugation, specimens in PAXgene tubes had significantly fewer DAPI negative cells than those from Streck tubes, but no difference was noted in cells obtained by density gradient centrifugation or when processing was delayed. The distribution of cell types was very similar among tube types in blood processed by density gradient centrifugation without storage but differed among tube types when processed by centrifugation. Fewer changes in the cell profile were observed when blood was stored for 24 h in EDTA or Streck BCT rather than Roche or PAXgene tubes before processing by density gradient centrifugation. Processing by centrifugation resulted in a much higher percentage of granulocytes than was observed after density gradient centrifugation and larger changes when processing was delayed.
Biospecimens
Preservative Types
- PAXgene
- Other Preservative
- None (Fresh)
- Streck/BCT
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Cell count/volume Hematology/ auto analyzer Cell count/volume Flow cytometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution Streck cell-free DNA BCT CE
Roche cell-free DNA collection tube
KEDTA tube
PAXgene Blood ccfDNA tubes
Storage Storage duration 0 h
24 h
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Biospecimen Aliquots and Components Blood processing method Centrifugation
Density gradient centrifugation
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Study Purpose
The purpose of this study was to determine if cells and cfDNA can be isolated simultaneously from EDTA blood. Peripheral blood from an unspecified number of healthy donors was spiked with 0.1% or 3.8% STA-NB-8 cells or with 15.9% of STA-NB-10 cells and 50 ng/mL fragmented DNA from neuroblastoma cell lines. Blood was processed after 0 and 24 h at 4°C both by centrifugation at 1600 x g for 10 min at 4°C to obtain plasma and by density gradient centrifugation using Lymphoprep. ctDNA was extracted from plasma using the QIAamp Circulating Nucleic Acid Kit and tumor cells were obtained from the mononuclear fraction by FACS sorting. Sequencing libraries were prepared with the NEBNext UltraTM II DNA Library Prep Kit for Illumina and sequenced on an Illumina HiSeq 3000 with single-end and 50bp or 65bp reads.
Summary of Findings:
Copy number profiles and data quality as well as NGS library yield and duplication rates were comparable in plasma isolated by standard double centrifugation and by density gradient centrifugation. Further, copy number profiles and NGS data were unaffected by storage for 24 h at 4°C. Copy number profiles including MYCN amplification in both cell lines, loss of chromosome 10 in STA-NB-8 cells, and loss of chromosome 6q in STA-NB-10 cells were determined from as few as 0.1% tumor cells. Importantly, the sensitivity for detection of copy number changes was higher using the cellular fraction than cfDNA.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Next generation sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Blood and blood products Plasma
Peripheral blood mononuclear cells
Storage Storage duration 0 h
24 h
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Biospecimen Aliquots and Components Blood processing method Centrifugation
Density gradient centrifugation