Effect of multiple plasma freeze-thaw cycles on non-invasive prenatal testing

Aim . To assess non-invasive prenatal testing (NIPT) as an informative criterion for quality of blood plasma and cell-free deoxyribonucleic acid (DNA) (cfDNA) in the case of using stabilization tubes at the preanalytical phase, in the example of plasma samples subjected to long-term storage at room temperature (+18о С) and multiple freezethaw cycles. Material and methods . The plasma samples were subjected to 20 freeze-thaw cycles (-80о С/+18о С), 20-day storage at +18о С with an intermediate cfDNA assessment. The quantitative yield was assessed by fluorometry, while the fragmentation and NIPT data — using realtime polymerase chain reaction (PCR) and high-throughput sequencing, respectively. Results. After multiple freeze-thaw cycles and long-term plasma storage at room temperature (+18о С), a decrease in the integrity and the concentration of cfDNA by the 20th thawing cycle, as well as a tendency to an increase in concentration by 20 days of storage were observed. Despite this, the NIPT results of the studied samples showed a high degree of coincidence with the NIPT data of the reference samples. Conclusion. The mere fact of successful NIPT cannot be considered as a reliable and sufficient criterion for assessing the quality of initial plasma and correct preanalytics. This emphasizes the particular importance of monitoring the conditions for transporting and storing plasma and whole blood samples.