Diverse fragment lengths dismiss size selection for serum cell-free DNA: a comparative study of serum and plasma samples.

Background: The objective of this study was to determine the features of fragment length for circulating cell-free DNA (cfDNA) from plasma and serum samples. Methods: Plasma and serum samples from different sources were randomly collected. Circulating cfDNA was extracted and purified by a precipitation-enriched and spin-column-based kit. The concentration of the purified DNA was immediately measured by a highly sensitive dsDNA quantitative assay, and then the fragment length was analyzed by capillary electrophoresis. The abundance of a specific fragment was estimated by the area under curve (AUC) for the fragment peak in the capillary electrophoresis. Results: A total of 199 plasma and 117 serum samples were extracted and analyzed. The average yield of cfDNA from the serum samples (131.67 ng/mL) was significantly higher than that from the plasma samples (32.78 ng/mL, p <0.001). The average abundance of the 20-400 bp fragments in plasma cfDNA (84.4%) was significantly higher than that of serum cfDNA (51.9%, p <0.001). Fragment peaks in serum cfDNA always presented in regions around 190 bp, 430 bp, and 630 bp, but plasma cfDNA generally showed a sharp peak in the 165-190 bp region and a much lower peak in the 300-400 bp region. Large fragments in plasma cfDNA were longer than 1000 bp and peaked around the 3000-4000 bp region while the large fragments in serum cfDNA were always shorter and peaked around the 1000 bp region. Conclusions: The fragment lengths of serum cfDNA and plasma cfDNA have very different features. Fragment size selection is suitable for plasma cfDNA but may not apply to serum cfDNA.