Comprehensive Evaluation of the Factors Affecting Plasma Circulating Cell-Free DNA Levels and Their Application in Diagnosing Nonsmall Cell Lung Cancer.
Author(s): Chen Z, Zhang S, Li C, Xu C, Zhao J, Miao L
Publication: Genet Test Mol Biomarkers, 2019, Vol. 23, Page 270-276
PubMed ID: 30986099 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of delayed centrifugation and plasma isolation protocol on white blood cell contamination and of cfDNA extraction method and freeze-thaw cycling of blood, plasma, and extracted DNA on the recovery of spiked-in DNA of different sizes. Differences in cfDNA yield based on patient diagnosis were also investigated.
Conclusion of Paper
Significant increases in the number of copies of hTERT in plasma were noted when blood was stored at 4˚ C for 48 h or at 25˚C for 24 h or more, indicating an increase in white blood cell lysis. Similarly, white blood cell contamination as determined by copies of hTERT decreased when a second centrifugation step was used and as speed or duration of the second centrifugation step increased. Recovery of DNA fragments varied with extraction kit and fragment length but overall was highest using the Magen HiPure Blood DNA Mini Kit. Freeze-thaw cycling of plasma or DNA in elution buffer had no effect on the recovery of spiked-in DNA, but freeze-thaw cycling of blood resulted in decreasing recovery of spiked-in DNA. Levels of cfDNA were comparable in patients with benign lung conditions and those with NSCLC but were significantly higher in patients with stage III/IV lung cancer than those with stage I/II.
Studies
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Study Purpose
This study investigated the effects of cfDNA extraction kit and freeze-thaw cycling on the recovery of spiked-in DNA of different lengths from blood and plasma. Blood and plasma from an unspecified number of healthy individuals were spiked with fragments of XT DNA of different lengths (92, 173, 249, and 312 bp). To evaluate the effects of extraction kit, cfDNA was extracted from spiked plasma in triplicate using the QIAamp DNA Blood Mini Kit, the QIAamp Circulating Nucleic Acid Kit, the Transgene EasyPure Viral DNA/RNA Kit, the Magen HiPure Blood DNA Mini Kit, and the Simgen Circulating Nucleic Acid Kit. The effect of freeze-thaw cycling was investigated using blood, plasma, and aqueous elution buffer spiked with fragments of XT DNA and compared to freshly prepared specimens. Copies of the spiked-in XT DNA were quantified by real-time RT-PCR.
Summary of Findings:
As expected, recovery of the fragments varied with extraction kit and fragment length. Recovery of the 92 bp fragments was highest with the Simgen Circulating Nucleic Acid Kit (83.5%) followed by the Magen HiPure Blood DNA Mini Kit (80.1%). Recovery of fragments ≥173 bp fragments was highest using Magen HiPure Blood DNA Mini Kit (95.5% for 173 bp, 91.9% for 249 bp, and 86.2% for 312 bp) followed by the QIAamp Circulating Nucleic Acid Kit for the 173 bp (88.8%) and 249 bp (90.2%) fragments and the Simgen Circulating Nucleic Acid Kit for the 312 bp fragment (80.3%). Recovery was lowest using the QIAamp DNA Blood Mini Kit (13.4-31.7%) followed by the Transgene EasyPure Viral DNA/RNA Kit (60.1-77.5%), regardless of fragment length. The recovery of cfDNA was unaffected by freeze-thaw cycling of plasma or cfDNA in extraction buffer but declined significantly with freeze-thaw cycling of blood (150.2% for 0 cycles, 83.8% for 1 cycle, 76.6% for 2 cycles, 66.9% for 3 cycles, and 69.0% for 4 cycles; P<0.05).
Biospecimens
Preservative Types
- None (Fresh)
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Blood and blood products Whole blood
Plasma
Biospecimen Preservation Type of fixation/preservation Frozen
None (fresh)
Analyte Extraction and Purification Analyte isolation method QIamp DNA Blood Mini Kit
QIAamp Circulating Nucleic Acid Kit
Transgene EasyPure Viral DNA/RNA Kit
Magen HiPure Blood DNA Mini Kit
Simgen Circulating Nucleic Acid Kit
Storage Freeze/thaw cycling 0 cycles
1 cycle
2 cycles
3 cycles
4 cycles
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Study Purpose
The purpose of this study was to investigate the effects of delayed centrifugation and centrifugation speed on white blood cell contamination of recovered cfDNA. Differences in cfDNA levels based on patient diagnosis were also investigated. Blood was collected from 64 patients with benign lung conditions (sarcoidosis, pneumonia, or other lung disease) and 74 patients with non-small cell lung cancer (NSCLC) into 5 mL EDTA tubes. Unless otherwise specified, plasma was obtained within 2 h of blood collection by centrifugation at 4˚C at 820 x g for 10 min followed by 25,000 x g for 10 min. The effects of storing whole blood at 4˚C and 25˚C for 1, 2, 4, 12, 24, and 48 h before processing was investigated in an unspecified number of specimens. To investigate the effects of centrifugation protocol, whole blood from an unspecified number of patients was centrifuged at 820 x g for 10 min without a second centrifugation step (1) at 820 x g for 10 min followed by 15,000 x g for 10 min (2), 20,000 x g for 10 min (3), or 25,000 x g for 10 min (4), at 820 x g for 5 min followed by 20,000 x g for 5 min (5), and at 820 x g for 20 min followed by 20,000 x g for 20 min (6). To investigate the effects of centrifugation speed on cfDNA recovery, plasma from healthy individuals was spiked with fragments of XT DNA and centrifuged at 820 x g for 10 min, 20,000 x g for 10 min, and 25,000 x g for 10 min. Plasma was frozen at -80˚C and cfDNA was extracted in triplicate using the Magen HiPure Blood DNA Mini Kit. hTERT was quantified by real-time RT-PCR.
Summary of Findings:
Significant increases in the number of copies of hTERT in plasma were noted when blood was stored at 4˚ C for 48 h or at 25˚C for 24 h or more, indicating an increase in white blood cell lysis. Similarly, hTERT levels were higher in specimens subjected to a single centrifugation at 820 x g than when two centrifugation steps were used. As speed or duration of the second centrifugation step increased, the number of copies of hTERT declined, indicating better removal of white blood cells. However, there was no effect of centrifugation speed on the recovery of spiked-in XT DNA from plasma, indicating that cfDNA itself is not affected by centrifugation speed. Levels of cfDNA were comparable in patients with benign lung conditions and those with NSCLC, but were significantly higher in patients with stage III/IV lung cancer than those with stage I/II (P=0.0339).
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
- Pneumonia/Respiratory Infection
- Other diagnoses
Platform:
Analyte Technology Platform DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage temperature 4˚C
25˚C
Storage Storage duration 0 h
1 h
2 h
4 h
12 h
24 h
Preaquisition Diagnosis/ patient condition Sarcoidosis
Pneumonia
NSCLC
Stage III/IV lung cancer
Stage I/II lung cancer
Real-time qPCR Specific Targeted nucleic acid hTERT
Biospecimen Aliquots and Components Centrifugation Different number of centrifugation steps compared
Multiple speeds compared
Multiple durations compared