Qualitative and quantitative comparison of cell-free DNA and cell-free fetal DNA isolation by four (semi-)automated extraction methods: impact in two clinical applications: chimerism quantification and noninvasive prenatal diagnosis.
Author(s): Pedini P, Graiet H, Laget L, Filosa L, Chatron J, Cherouat N, Chiaroni J, Hubert L, Frassati C, Picard C
Publication: J Transl Med, 2021, Vol. 19, Page 15
PubMed ID: 33407582 PubMed Review Paper? No
Purpose of Paper
This paper compared the concentration and integrity of isolated cell-free DNA (cfDNA) as well as the detection of chimerisms and RhD status in cfDNA isolated from plasma using four different automated extraction methods. Different methods of DNA quantification and chimerism detection were also compared.
Conclusion of Paper
The Qubit-quantified concentration of DNA was significantly higher when extraction was performed using the IDEAL or LABTurbo 24 automated systems compared to the MagNA Pure or CHemagic 360 systems but no significant differences in DNA concentration among methods were found when quantification was with ddPCR or BIAbooster. The size of the first capillary electrophoresis peak was significantly smaller and the percentage of DNA 75-239 bp higher when extraction was with the MagNA Pure 24 rather than any of the other methods. The chimerism (male DNA spiked into female plasma) was not quantifiable by ddPCR but 8 of 24 chimerisms were quantifiable using next-generation sequencing (NGS) when DNA was extracted using the LABTurbo system. cfDNA isolation method had no effect on the detection of fetal Rhesus D (RhD) status. DNA concentration was dependent on the measurement method but was modestly correlated between Qubit HS and ddPCR.
Studies
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Study Purpose
This study compared the concentration and integrity of isolated cfDNA as well as the detection of chimerisms and RhD status in cfDNA isolated from plasma using four different automated extraction methods. Different methods of DNA quantification and chimerism detection were also compared. Blood was collected into Roche Cell-free DNA collection tubes from one male and one female healthy patient and 20 pregnant RhD negative women with known RhD fetal status. Plasma was obtained by room temperature centrifugation at 1600 x g for 10 min followed by 4500 x g for 10 min. Chimerism plasma was constructed by spiking 10% and 1% plasma from the male patient into the female patient’s plasma. DNA was extracted from 2 mL plasma using the Roche MagNA Pure 24 Total NA Isolation Kit, the Perkin Elmer Chemagic 360 NextPrep-Mag cfDNA Isolation Kit, the IDSolution IDEAL IDXTRACT-MAG Kit, and the LABTurbo 24 using a Virus Combo Kit and stored at -20°C until use. cfDNA was quantified by ddPCR amplification of RPP30, Qubit dsDNA HS assay, and the capillary electrophoresis-based BIABooster. Chimerism was quantified by ddPCR amplification of a region of the Y chromosome and RPP30 and by next-generation sequencing using the Devyser Chimerism Kit. RhD status was detected using the real-time PCR DNA Fetal Kit RhD.
Summary of Findings:
The Qubit-quantified DNA concentrations were significantly higher when extraction was performed using the IDEAL or LABTurbo 24 automated systems rather than the MagNA Pure or Chemagic 360 systems (P=0.01, all), but no significant differences in DNA concentration among methods were observed when quantification was with ddPCR or BIAbooster. Measurement of DNA concentrations were significantly different using the Qubit HS method compared to ddPCR or the capillary electrophoresis-based BIAbooster (P=0.01, both), but results were statistically comparable when quantification was with ddPCR and BIAbooster (P=0.78). Quantified concentrations were correlated between Qubit HS and ddPCR (rs=0.50, P=0.024) but not between BIAbooster and ddPCR or Qubit. The size of the first capillary electrophoresis peak was significantly smaller when extraction was with the MagNA Pure 24 compared to the other methods (93-129 bp versus 163-167 bp, P<0.005) with an average size of 75-239 bp for 90% of the DNA and 240-369 bp for 5% of the DNA compared to 71-79% (P=0.009) and 15% (P<0.005), respectively, using the other methods but no difference in percentage of DNA 370-579 and 580-1649 bp. The chimerism (male DNA spiked into female plasma) was not quantifiable by ddPCR. Using NGS, 8 of 24 chimerisms were quantifiable in DNA extracted using the LABTurbo system, but the chimerisms were either not detected or detected at less than 1000 X using the other isolation methods. All three RHD exons were detected at comparable cycle threshold values in plasma from pregnant women carrying a RhD+ fetus and not in those carrying a RhD- fetus, regardless of cfDNA isolation method. However, CT values for the exogenous DNA were lower when extraction was with LABTurbo and IDEAL than the other methods.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Pregnant
- Normal
Platform:
Analyte Technology Platform DNA Automated electrophoresis/Bioanalyzer DNA Digital PCR DNA Next generation sequencing DNA Fluorometry DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method MagNA Pure 24
IDEAL
LABTurbo 24
Chemagic 360
Digital PCR Specific Technology platform Qubit dsDNA HS Assay Kit
BIAbooster,
Next generation sequencing Specific Technology platform ddPCR