Cell-free DNA (CFDNA) is a cell-free DNA segment that is widely present in human plasma, cerebrospinal fluid, urine, pleural fluid, ascites, or feces. The concentration of CFDNA in the peripheral blood of healthy people is mostly less than 100 ng ml, and the average is about 30 ng ml. CFDNA levels may be elevated in disease states (e.g., tumors, postoperative, viral infections, cardiovascular diseases, etc.). CFDNA studies have received a lot of attention because they can be used to screen biomarkers for liquid biopsies and other clinical research applications. Plasma CFDNA research has promoted the development of non-invasive prenatal diagnosis (NIPT) testing technology, and the advantages of CFDNA testing include non-invasiveness, low risk, and high accuracy. Therefore, CFDNA has great potential application value in the early diagnosis, prognosis, and monitoring of pregnancy or clinical diseases.
1. On March 1, 2018, the joint research results of Cerba Laboratory in Paris, France and other units were published in the journal Genetics in Medicine (Impact Factor 8822) entitled "Cell-free fetal DNA versus maternal serum screening for trisomy 21 in pregnant women with and without assisted reproduction technology: a prospective interventional study". In this prospective interventional study, scientists performed cell-free DNA (CFDNA) sequencing and maternal serum screening (MSS) in pregnant women conceived naturally (SP) or by assisted reproductive technology (ART) to screen for the onset of trisomy 21.
Background: CFDNA has been used for several years as a preliminary screening test, but few studies have been conducted in a prospective manner** on this option. It has been reported that the MSS program has a lower detection accuracy than SP for pregnancies with the help of ART, so this issue is worth paying attention to.
Methods: This prospective interventional study was conducted to study the performance of CFDNA and MSS in pregnancy with or without Art. Each volunteer underwent MSS and CFDNA testing to screen for fetal trisomy 21. A total of 794 volunteers were enrolled in the preliminary analysis cohort, including 472 volunteers in the SP group and 322 pregnant volunteers after antiretroviral**. In this study, the Refdi's Labchip GX nucleic acid analyzer was used to perform quality control analysis of CFDNA-sequencing libraries.
Results: The false positive rate and positive ** value of MSS were 6., respectively, compared with the screening results of two methods for the detection of fetal trisomy 216% and 88%, compared to 0% and 100% for cfdna, respectively. The MSS false-positive rate and positive ** values in the ART group were 11., respectively7% and 26% and 3 in the SP group2% and 211%。When using CFDNA testing, the global incidence of invasive procedures is 19% (15 794), compared to 8 if MSS is used alone4%（65/794）。
Table 1Comparison of the detection performance of MMS and CFDNA for trisomy 21 in SP group and ART pregnancy group.
Conclusions: Overall, the screening results of fetal trisomy 21 indicate that the screening performance of CFDNA for trisomy 21 is significantly better than that of MSS. CFDNA outperformed MSS in both SP and ART pregnancies, reducing the number of invasive procedures. The results of this study suggest that CFDNA should be considered as a primary clinical screening modality, particularly in pregnancy screening obtained after antiretroviral**.
Second, on March 12, 2018, the collaborative research results of Life&Soft, AP-HP and Cerba laboratories in France were published in the journal Scientific Reports (Impact Factor 4379) entitled "Using massively parallel shotgun sequencing of maternal plasmatic cell-free DNA for cytomegalovirus DNA detection during pregnancy: a proof of concept study". This is a proof-of-concept study for the detection of cytomegalovirus (HCMV) DNA by massively parallel sequencing of maternal plasma CFDNA.
Background: HCMV infection in pregnant women can lead to congenital infection of the fetus, which will have a serious impact on the health of newborns. Studies have shown that 1000 100 billion DN** segments per milliliter of plasma are circulating cell-free. At present, the research of this type of DNA has been extended to the field of NIPT applications.
Methods: Shotgun sequencing (MPSS) was performed on 27 positive calibration samples and 58 negative plasma CFDNA samples to analyze the correlation between sequencing depth and viral load. Subsequently, a confirmatory analysis of HCMV virus detection was performed in a population cohort of 538 pregnant women, of which 36 were sequenced twice, resulting in a total of 574 pregnant women's plasma CFDNA data.
In this study, Perkinelmer's LabChip GX Nucleic Acid Analyzer was used for quality control analysis of CFDNA-sequencing libraries.
Results: This study demonstrated that specific readouts of the virus could be detected in NIPT data from massively parallel shotgun sequencing. In addition, there is a strong correlation between the viral load of the calibration sample and the number of reads aligned on the reference genome. Based on the above results, the research team constructed a statistical model that was able to quantify the viral load of patient samples.
Figure 1Review of HCMV sequencing results in a cohort of pregnant women.
a) Summary of raw sequencing results from 538 plasma samples;(b) The average depth of HCMV samples in 538 plasma samples;(c) Perform a detailed comparison of reads on positive samples.
Conclusions: It is recommended to use this new method to detect and quantify circulating DNA viruses (e.g., HCMV) during pregnancy, which can be used to improve the diagnostic level of NIPT.
References: 1 costa jm, letourneau a, f**re r, et al. cell-free fetal dna versus maternal serum screening for trisomy 21 in pregnant women with and without assisted reproduction technology: a prospective interventional study. genet med. 2018, 20(11):1346-1353. doi: 10.1038/gim.2018.4.
2. chesnais v, ott a, chaplais e, et al. using massively parallel shotgun sequencing of maternal plasmatic cell-free dna for cytomegalovirus dna detection during pregnancy: a proof of concept study. sci rep. 2018, 8(1):4321. doi: 10.1038/s41598-018-22414-6.
Labchip GX Nucleic Acid Analyzer—Less sample consumption, more sensitivity.
Labchip GX Touch Nucleic Acid Analyzer.
The microfluidic capillary electrophoresis separation technology can quickly complete the detection of the size and concentration of DNA and RN** segments, and provide high-throughput and accurate nucleic acid quantification—the lower limit of detection concentration reaches 05 pg/μl。
Depending on the sample throughput, you can choose an efficient platform with a batch throughput of 24 samples or a model with a maximum batch throughput of 384 samples for your high-throughput workflow.
The convenient touchscreen interface allows even novices to quickly prepare and run samples, simplifying many of the tedious steps of the traditional electrophoresis process.
Data output is further enhanced by data visualization software, and electronic data results generated in real-time can be immediately viewed, analyzed, or reported in the form of virtual glue plots, electrophoresis peak plots, or summaries**.
Figure 2Labchip GX Touch Nucleic Acid Analyzer.