The Effect of Freezing on Non-invasive Prenatal Testing

Plasma cryopreservation is unavoidable in China, due to technical specifications requiring storage of additional plasma at −80 degrees for three years. However, the effect of freezing on non-invasive prenatal testing (NIPT) is still uncertain. We collected 144 euploid pregnant samples, 22 on trisomy 21, 4 on trisomy 13, and 3 on trisomy 18, by massively parallel sequencing before and after freezing. Compared with the success rate of 100% of fresh samples, the detection success rates of trisomy 21, trisomy 13 and euploidy in frozen samples by NIPT were 95.45%, 75% and 95.14%, respectively. Of these, 9 cases of frozen sample sequencing failed, with 8 cases being due to high GC content. The chromosome 21 (chr21) z-value of the frozen trisomy 21 samples was lower than that of fresh samples. Meanwhile, freezing reduced the male positive foetal cell-free DNA (cfDNA) fraction, which was accompanied by an increase in the Unimap-GC level in the massively parallel sequencing data and a decrease in the Unique reads/Total reads ratio. Laboratory freezing reduced the chr21 z-value of foetal trisomy 21, which can be explained by a reduction in the foetal cfDNA fraction and effective Unique reads for NIPT analysis. The Unimap-GC content of the serum samples after freezing was higher, which can lead to failure of NIPT detection.