Feasibility of Droplet Digital PCR Analysis of Plasma Cell-Free DNA From Kidney Transplant Patients.
Author(s): Jerič Kokelj B, Štalekar M, Vencken S, Dobnik D, Kogovšek P, Stanonik M, Arnol M, Ravnikar M
Publication: Front Med (Lausanne), 2021, Vol. 8, Page 748668
PubMed ID: 34692738 PubMed Review Paper? No
Purpose of Paper
This paper assessed the potential impact of isolation method on cfDNA yield and variability from plasma. The effect of timing post-kidney transplant on cfDNA yield was also investigated.
Conclusion of Paper
The mean cfDNA yield from plasma was slightly higher when extraction was with the QIAamp CNA kit compared to MagMAX and MiniElute kits, but the differences were not statistically significant. Intersample variability in cfDNA yield was highest when the MagMAX kit was used but intrasample variability was highest when extraction was with the CNA kit. The fragment size profile was comparable among cfDNA isolated with each of the methods. The authors report, cfDNA yield measured by Qubit and ddPCR copy number for SNP detection were strongly correlated when cfDNA was isolated with the QIAamp CNA kit. Average cfDNA yield was higher in plasma from kidney transplant recipients than healthy volunteers but the difference was only significant for specimens collected within 14 days of transplant.
Studies
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Study Purpose
This study assessed the potential impact of isolation method on cfDNA yield and variability from plasma. Potential effects on cfDNA yield due to the timing of blood collection after kidney transplant were also investigated. Blood was collected from 10 healthy volunteers and 8 kidney transplant patients (4 patients 6 days post-transplant, and 4 patients at multiple timepoints <13 months post-transplant) into Streck Cell-Free DNA BCT and stored at room temperature <72 h. Plasma was obtained by centrifugation at 1,600 g for 10 min followed by 16,000 g at 10 min. Plasma was stored at -80°C and the buffy coat was stored at -20°C. cfDNA was extracted from plasma of healthy volunteers using the QIAamp Circulating Nucleic Acid Kit with the QIAvac 24 Plus Vacuum System, QIAamp MiniElute ccfDNA Kit, and the MagMAX Cell-Free DNA Isolation Kit. cfDNA was extracted from the plasma of kidney transplant recipients using the QIAamp Circulating Nucleic Acid Kit. Genomic DNA from buffy coat of kidney transplant patients was isolated with the QIAamp DNA Mini Kit and stored at -20°C. DNA yield was quantified using the Qubit dsDNA HS Assay Kit and fragment size determined using HT DNA High Sensitivity LabChip Kit and Caliper LabChip GX instrument. Genotype was analyzed in cfDNA isolated with the QIAamp CNA kit using the TaqMan Custom SNP Genotyping Assay on a QX200 AutoDG ddPCR System.
Summary of Findings:
The mean cfDNA yield from plasma was highest when extraction was with the QIAamp CNA kit (6.1 ng/mL), followed by the MagMAX kit (4.6 ng/mL), then the MiniElute kit (4.3 ng/mL), but differences were not statistically significant. Intersample variability was highest when cfDNA isolation was with the MagMAX kit (5.3-fold between highest and lowest versus 3.4-fold with CNA and 2.8-fold with MiniElute), but intrasample variability ranged from 13% for the MiniElute kit to 18% for the CNA kit. The fragment size profile was comparable among cfDNA isolated with each of the methods. The authors report, cfDNA yield measured by Qubit and ddPCR copy number for SNP detection were strongly correlated when cfDNA was isolated with the QIAamp CNA kit. The average cfDNA yield was higher in plasma from kidney transplant recipients than healthy volunteers (P=0.007). While cfDNA yield was significantly higher in plasma collected from kidney transplant patients ≤14 days post-transplant (P=0.006), differences were not significant when plasma was collected >14 days after transplant.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Digital PCR DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Diagnosis/ patient condition Healthy
Kidney transplant recipient
Fluorometry Specific Technology platform ddPCR
Analyte Extraction and Purification Analyte isolation method QIAamp Circulating Nucleic Acid Kit with the QIAvac 24 Plus Vacuum System
QIAamp MiniElute ccfDNA Kit
MagMAX Cell-Free DNA Isolation Kit
Biospecimen Acquisition Time of biospecimen collection ≤14 days post-transplant
>14 days post-transplant