Affiliation:
1. Department of Pathology Helios Klinikum Emil von Behring Berlin Germany
2. Department of Pneumology Helios Klinikum Emil von Behring Berlin Germany
3. Department of Pneumology Charité ‐ Universitätsmedizin Berlin Berlin Germany
Abstract
AbstractBackgroundUrine holds promise as a source for cell‐free DNA (cfDNA) analysis of cancer genetics due to its nature as a self‐collectable biospecimen available in large quantities. However, pre‐analytical variables such as preservation of cfDNA or efficiency of up‐scaling specimen volume need to be better explored to increase analysis sensitivity.Patients and MethodsInitially effects of pH levels on cfDNA stability of urine preserved with EDTA were investigated in three healthy probands. Furthermore, the efficiency of urine volume up‐scaling was examined using a simple DNA extraction method and cfDNA in urine of 32 individuals. Quantification was performed by PCR detection of three relevant targets used for EGFR and KRAS gene mutational analysis.ResultsOnly samples preserved with EDTA at alkaline pH levels showed cfDNA stability of up to 10 days at room temperature. Moreover, it was found that increasing the volume up to 100 mL was highly efficient. A similar amount of copies was detected in three different gene sites in all specimens indicating both a good availability and a non‐random distribution pattern across genes. Since the median cfDNA copy number was 1642 copies/mL, abundance of cfDNA in this type of liquid biopsy is low.ConclusionPredictable sensitivities with different urine volumes on the ground of detectable cfDNA in our study population revealed that up‐scaling (>5 mL) is mandatory if the mutation allele frequency is less than 1%.
Subject
Microbiology (medical),Biochemistry (medical),Medical Laboratory Technology,Clinical Biochemistry,Public Health, Environmental and Occupational Health,Hematology,Immunology and Allergy