The cfDNA is today a source of clinical biomarkers in blood sample. However, this utility is limited by its low concentration and small fragment size. The variations of these two characteristics of cfDNA inducing different analytical results depend on the isolation method used. Thus, in response to a tender, this study presents 4 high efficient isolation automats for cfDNA isolation. Three of the four extractors (LABTurbo 24, Chemagic 360, MagNA Pure 24) are CEIVD marked. They are all characterized by an extraction of a maximum of 24 tubes in a time varying from 45 minutes to 2 hours. Two automated systems (LABTurbo 24 and MagNA Pure 24) integrated full process traceability (from the primary tube to the elution).
The IDEAL and LABTurbo methods have a higher extraction yield by QUBIT HS than the two other isolation methods. Other authors who have previously shown that the extractors have different cfDNA extraction yields. Thus, Fleischhacker et al. (18) had compared extraction with Qiagen®, MagNa Pure® and NucleoSpin instruments from 44 samples and showed that the quantification of cfDNA measured by qPCR varied from 1.6 ng / mL to 28.1 ng / mL depending on the method used. Likewise, Perez-Barrios et al. (19) noted these variations by comparing MagNa Pure® and Maxwell®RSC on 26 samples, using the QUBIT 2.0 Fluorometer and ddPCR.
This study highlights that the 3 assay techniques give different concentrations of cfDNA for the same sample with large variations sometimes, especially between QUBIT and the two other methods. These effects have also been observed by others (20, 21). An explanation should be the difference in their ability to accurately quantify different fragment sizes of cfDNA. Also, compared with intact (unfragmented) genomic DNA, qPCR measurements showed a 67% reduction in the concentration for DNA fragments with a size of 150 bp, while PicoGreen measurements only showed a 29% reduction (22). In opposite, the amount of DNA measured by the NanoDrop instrument was not affected by fragmentation to 150 bp, probably because fragmentation does not affect absorbance measurements (23).
The cfDNA amounts measured with the QUBIT fluorometer and the ddPCR are quite comparable, but not with BIABooster. This discrepancy could be explained by RNAse I pretreatment before electrophoretic migration. Indeed, QUBIT HS uses a target-selective dye that emit fluorescence when bound to DNA and amplification dPCR mixes are DNA specific. However, the cfDNA concentrations quantified by ddPCR and QUBIT HS were not modify with RNAse I pretreatment (data not shown).
To the best of our knowledge, both different assays in this study have not yet been included in such a comparative study. Therefore, we cannot state with complete confidence that these different methods are more suitable than the other at quantifying short fragments of cfDNA. Also, these inter-method variations induce a single quantification method for an application in a laboratory. Likewise, they cause difficulties in comparing results between different laboratories, when different DNA concentration measurement methods are used.
Except to MagNa Pure method, other methods give a same cfDNA size profile with a first peak around 165 pB and second peak around 311 pB, the first representing a mean of 76% of cfDNA fragments. The MagNa Pure method isolates a larger small fragment with first peak around 119 pB, representing 90% of cfDNA fragmented. Another study showed that the MagNa Pure® provides a smaller amount of cfDNA with a larger amount of small cfDNA (150–200 bp) measured by Agilent 2100 Bioanalyzer (19). Kloten V et al. observed the differences obtained during extraction on a silica membrane or on magnetic beads. They noted that if the amounts of cfDNA are relatively similar, the size profile is different depending on the extraction technique used. Indeed, extraction on magnetic beads provides a size profile with a larger amount of small cfDNA fragments (< 600 bp) while extraction on a silica membrane provides a size profile with a larger quantity of fragments of large sizes (> 600 bp) characteristic of cfDNA originating from cells lysed during the extraction process (24). This ascertainment is not verified in our hand because LABTurbo isolates cfDNA on column extraction and the three others on magnetic beads.
The use of cfDNA in screening for rejection of kidney, heart, lung and pancreatic transplants has been published (8–13). These studies generally show a persistent increase in donor cfDNA detected in patients with biopsy-confirmed rejection. The donor cfDNA chimerism quantification techniques are either RQ-PCR or ddPCR and more recently NGS, with a sensitivity of each technique around 1%. Our study used the ddPCR and the NGS methods to achieve 10% and 1% chimerism, from male plasma diluted in maternal plasma. Surprisingly, only the LABTurbo method could detect the two percentages of chimerism and only by the NGS technique. An explanation is that this isolation method is one of the two methods that allows the extraction of a large amount of cfDNA when measured by QUBIT HS. Similarly, the difference in results between the two techniques of chimerism quantification can be explained by the ability of NGS to amplify shorter strands (70 bp) than ddPCR (200 bp). In our hand, their sensitivity of chimerism quantification from genomic DNA is similar (i.e 0.1%; data not shown). However, their sensitivity for the chimerism quantification from cfDNA in an organ transplant or other context has not been compared to this date. Furthermore, the plasma mixes are artificial and these results cannot fully reflect the behavior of cfDNA in a sample.
In context of NIPT, all methods allow the RHD cffDNA detection using the Free DNA Fetal Kit® RhD. However, the Ct of the exogenous DNA and the various RHD exons are generally lower for the LABTurbo and IDEAL methods than for the others, suggesting a better cfDNA extraction yield for these two methods and inducing probably a higher sensitivity of RHD cffDNA detection. The small discrepancy between the Ct values of three RHD exons, not exceeding 2 Ct values seems not to depend on isolation methods. It has been suggested than the variation of plasma preparation protocols or the low concentration of cffDNA in mothers’ plasma can induce this difference (25).
Limitations of this study are primarily the small sample numbers, which probably limited the power to observe different effects. The extractions were performed only in two runs, after installation and qualification of the automated system in the laboratory. Also, this study presents assays results without optimization of each isolation method. Each supplier tested only one reagent kit on their instrument; some may offer others. The evaluation of the detection of RHD-cffDNA used different plasmas for each isolation method. Likewise, the weeks of amenorrhea of the women sampled are quite different for each method.