Validating quantitative PCR assays for cell-free DNA detection without DNA extraction: Exercise induced kinetics in systemic lupus erythematosus patients

Circulating cell-free DNA (cfDNA) has been investigated as a screening tool for many diseases. To avoid expensive and time-consuming DNA isolation, direct quantification PCR assays can be established. However, rigorous validation is required to provide reliable data in the clinical and non-clinical context. Considering International Organization for Standardization, as well as bioanalytical method validation guidelines we provide a comprehensive procedure to validate assays for cfDNA quantification from unpurified blood plasma. A 90 and 222 bp assay was validated to study the kinetics of cfDNA after exercise in patients with systemic lupus erythematosus. The assays showed ultra-low limit of quantification (LOQ) with 0.47 and 0.69 ng/ml, repeatability [≤] 11.6% (95% CI: 8.1-20.3), and intermediate precision [≤] 12.1% (95% CI: 9.2-17.7). Incurred sample reanalysis confirmed the precision of the procedure. The additional consideration of pre-analytical factors shows that centrifugation speed and temperature do not change cfDNA concentrations. In SLE patients cfDNA increases [~]2 fold after all out walking exercise, normalizing after 60 min of rest. The established assays allow reliable and cost-efficient quantification of cfDNA in minute amounts of plasma in the clinical setting and can be used as a standard to control pre-analytical factors including cfDNA losses during purification.