A novel method for extracting circulating cell-free DNA from whole blood samples and its utility in the non-invasive prenatal test.

OBJECTIVE: We verified a magnetic bead-based, simple, and fast method for circulating cell-free DNA (cfDNA) extraction from whole blood samples(CEWB) and characterised its utility in non-invasive prenatal testing (NIPT). METHOD: We extracted cfDNA from both plasma and whole blood of the patients using CEWB and compared it to that extracted using a Qiagen extraction kit; droplet digital polymerase chain reaction test was used to calculate the fragment size bias. In all, 304 samples were used for NIPT. RESULTS: The CEWB group (mean ± standard deviation [SD]: 4.34 ± 0.41 ng/ml plasma) reported less DNA weight yield than the Qiagen group (4.90 ± 0.50 ng/ml plasma). There was no significant difference between the CEWB group and the Qiagen group in the gene fragments (136 bp: p = 0.064 and 420 bp: p = 0.534). In a parallel cohort study to characterise the utility of the CEWB method in NIPT, the treatment group extracted by CEWB showed a sensitivity of 100%, a specificity of 99.65%, and a positive predictive value of 95%. CONCLUSIONS: This study demonstrated that CEWB achieves an acceptable yield of DNA without contamination from genomic DNA. Subsequent clinical experiments in a parallel cohort indicated its utility for NIPT.

The effect of hemolysis on quality control metrics for noninvasive prenatal testing.

BACKGROUND: Noninvasive prenatal testing (NIPT) is the testing of blood samples from pregnant women to screen for fetal risk of chromosomal disorders. Even though in vitro hemolysis of blood specimens is common in clinical laboratories, its influence on NIPT has not been well investigated. METHODS: Peripheral blood samples were collected from 205 pregnant women and categorized according to the concentration of free hemoglobin in the plasma. After performing NIPT using massively parallel sequencing, the quality control metrics were analyzed and compared with samples that did not undergo hemolysis or samples redrawn from the same women. RESULTS: The specimens were divided into four groups based on the concentration of free hemoglobin: Group I (0-1 g/L, n = 53), Group II (1-2 g/L, n = 97), Group III (2-4 g/L, n = 30), and Group IV (> 4 g/L, n = 25). There was no significant difference in the quality control metrics of clinical samples with slight or moderate hemolysis (Group II and III). However, samples with severe hemolysis (Group IV) showed a significantly increased rate of duplicated reads (duplication rate) and fetal fraction, as well as decreased library concentration compared with samples without hemolysis. Moreover, the increase in fetal fraction caused by hemolysis was confirmed by redrawing blood samples in Group IV. CONCLUSION: For NIPT using massively parallel sequencing, samples with slight or moderate hemolysis (≤ 4 g/L) are acceptable. However, careful consideration should be taken regarding the use of severely hemolyzed samples (> 4 g/L), since they might increase the risk of test failure.