Investigation of preanalytical variables impacting pathogen cell-free DNA in blood and urine

Pathogen cell-free DNA (pcfDNA) in blood and urine is an attractive biomarker; however, the impact of preanalytical factors is not well understood. Blood and urine samples from healthy donors spiked with cfDNA from Mycobacterium tuberculosis, Salmonella enterica, Aspergillus fumigatus and EBV, and samples from tuberculosis patients were used to evaluate the impact of blood collection tube, urine preservative, processing delay, processing method, freezing and thawing, and sample volume on pcfDNA. PCR cycle threshold (CT) was used to measure amplifiable cfDNA. In spiked samples, median CT for M. tuberculosis, S. enterica, and EBV cfDNA was significantly lower in blood collected in K2EDTA than Streck and PAXgene blood collection tubes, and significantly lower in EDTA-urine than Streck-urine. Blood and urine samples from TB patients preserved with K2EDTA and Tris-EDTA, respectively, showed significantly lower median M. tuberculosis CT compared with Streck blood collection tube and urine preservative. Processing delay increased median pathogen CT for Streck and PAXgene but not K2EDTA blood samples, and for urine preserved with Streck reagent but not EDTA. Double spin compared with single spin plasma separation increased median pathogen CT regardless of blood collection tube. No differences were observed between whole urine and supernatant, and between fresh and thawed plasma and urine after 24 weeks at -80 °C. Larger plasma and urine volume in contrived and patient samples showed a significantly lower median M. tuberculosis CT. These findings suggest large volume single spin K2EDTA-plasma and EDTA-whole urine with up to 24-hour processing delay may optimize pcfDNA detection.