Analytical performance of cfDNA quantification methods in pediatric acute lymphoblastic leukemia plasma.

OBJECTIVES

Cell-free DNA (cfDNA) is a highly regarded noninvasive biomarker in cancer diagnosis, but analytical variability may hinder its practical application. This study aims to assess the analytical performance of various cfDNA measurement techniques using plasma samples from children with acute lymphoblastic leukemia (ALL).

METHODS

Plasma was obtained from 1500 pediatric patients with ALL between the ages of 4 and 13 years. Standardized methods for obtaining double-centrifuged plasma were used to reduce the possibility of contaminating genomic DNA. The extraction of cfDNA was done by a commercial kit using a silica membrane technique and optimized phenol-chloroform-isoamyl alcohol approach. Determination of concentration was done by fluorimetry, spectrophotometry, and enzyme-linked immunosorbent assay. Detection of cfDNA was done using PCR of PTEN gene.

RESULTS

Concentrations of cfDNA were found to be significantly lower when determined using fluorometry compared to spectrophotometry with a higher level of consistency, implying that spectrophotometric detection was nonspecific. ELISA-based detection demonstrated relatively consistent signal generation across samples; however, it represents an indirect approach to cfDNA quantification. Manual extraction was associated with a higher concentration of cfDNA, while the kit method produced purified cfDNA samples suitable for PCR amplification.

CONCLUSION

A high level of influence of analytical methodology is evident regarding the results of cfDNA quantitation in the blood plasma of patients with pediatric ALL.