Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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The art of obtaining a high yield of cell-free DNA from urine.

Author(s): Augustus E, Van Casteren K, Sorber L, van Dam P, Roeyen G, Peeters M, Vorsters A, Wouters A, Raskin J, Rolfo C, Zwaenepoel K, Pauwels P

Publication: PLoS One, 2020, Vol. 15, Page e0231058

PubMed ID: 32251424 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to investigate the effects of collection timing, preservation, storage duration and temperature, and centrifugation protocol on the yield of DNA and the cfDNA to gDNA ratio in urine specimens. DNA yields were measured by multiple methods. Urine was collected between 8 AM and 6 PM from 39 healthy volunteers and 14 patients with colorectal cancer, pancreatic ductal adenocarcinoma, breast cancer, or non-small cell lung cancer with metastases. To investigate the effect of collecting first void versus random timing, urine was collected from five healthy volunteers and two cancer patients in two consecutive fractions. To investigate the effect of collection time, three to five urine specimens were collected from four healthy volunteers at varying timepoints throughout the day. To investigate the effects of stabilizers, urine specimens from five healthy volunteers were used for immediate analysis, aliquoted, and then analyzed after 1 week of storage at room temperature without preservative or when preserved with Streck cfDNA Urine Preserve, an in-house urine conservation medium, or ThinPrep Cytolyt Solution. To investigate the effects of storage time and temperature, urine from four healthy volunteers and two cancer patients was analyzed immediately and again after 2, 18, 24, 48, 72, and 96 h at room temperature or 4°C. To investigate the effects of storage time and temperature on Streck-preserved urine, urine from four healthy volunteers was mixed with Streck cfDNA Urine Preserve and stored for 18, 24, 48, and 72 h at 4°C, room temperature, or 30°C. To investigate the effects of urine preparation method, urine from three healthy volunteers was aliquoted and centrifuged at: 1) 750 x g for 10 min followed by 2680 x g for 10 min, 2) 750 x g for 10 min followed by 3350 x g for 15 min, 3) 750 x g for 20 min followed by 2680 x g for 10 min, 4) 750 x g for 20 min, or 5) 500 x g for 30 min. To investigate the effects of urine preparation method, urine from four healthy volunteers and four cancer patients was preserved with Streck cfDNA Urine Preserve, aliquoted, and centrifuged at: 1) 4000 x g for 10 min, 2) 3000 x g for 15 min, 3) 2680 x g for 10 min, or 4) 750 x g for 10 min. Unless otherwise specified, urine specimens were centrifuged at 750 x g for 15 min and cfDNA was extracted using the Quick-DNA Urine Kit. cfDNA was stored at -20°C and quantified using the Qubit dsDNA High Sensitivity Assay Kit and a Qubit 3.0 Fluorometer, the droplet digital PCR-based KRAS G12/G13 Screening Multiplex Kit, and the DNF-474-33 –High Sensitivity NGS Fragment Analysis Kit on an Applied Biosystems Fragment Analyzer.

   Summary of Findings:

   The first void contained 28%- to >80% of isolated DNA when analyzed by fluorometry or ddPCR, with approximately 50% or more of the DNA obtained in the first void from 6 of the 7 patients.  Similarly, when analyzed by fragment analyzer, more than 50% of the DNA was obtained from the first void in all specimens, but the exact percentages and rank order differed. In a second study, it was found by fluorometry and ddPCR that the highest concentration of DNA was found in the second or third void rather than the first void, but void 1, 4 and 5 had the highest cfDNA to gDNA ratio. The amount of DNA obtained decreased when a second specimen was obtained within 1.5 h after the prior collection and increased when obtained more than 3.5 h after previous void. Storage of urine for 1 week without preservation or when preserved with Cytolyt resulted in loss of most of the DNA but this loss was mostly attenuated when preserved with an in-house UCM or the Streck urine preservative. Fragment analysis showed a higher percentage cfDNA in specimens preserved in Streck urine preservative rather than UCM. Storage of urine at 4°C stabilized the DNA concentration for 96 h as determined by fluorometry and had no further effect after 2 h as measured by ddPCR. In contrast, storage at room temperature beyond 18 h resulted in increased measured DNA as determined by fluorometry and variable levels as determined by ddPCR. While the cfDNA to gDNA ratio increased very slightly with urine storage at 4°C, it declined rapidly and was variable with room temperature storage. Importantly, KRAS mutations were detected in urine specimens after storage at room temperature for up to 96 h. In contrast to unpreserved urine, DNA concentration (ddPCR and fluorometry) in Streck-preserved urine were stable at room temperature or 30°C for up to 72 h but declined when stored at 4°C. However, the cfDNA to gDNA concentration was unaffected by storage temperature. The highest total DNA yields were obtained when centrifuged at 500 x g followed by a single step at 750 x g and the lowest when centrifuged at 750 x g for 10 or 20 min followed by at 2,680 x g or 3,350 x g. Not surprisingly, the ratio of cfDNA to gDNA was higher in specimens centrifuged at 750 x g for 10 or 20 min followed by at 2,680 x g or 3,350 x g than those centrifuged just once at 500 or 750 x g. In Streck-preserved urine, centrifugation at 4,000 x g for 10 min or 3,000 x g for 15 min was deemed the best as they resulted in a lower DNA concentration and higher cfDNA to gDNA ratio. Importantly, the PIK3CA mutations were detected in specimens from the one patient harboring this mutation, regardless of centrifugation protocol, and KRAS mutations were detected in urine specimens after storage at room temperature for up to 96 h.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • None (Fresh)
   • Other Preservative

   Diagnoses:

   • Normal
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Automated electrophoresis/Bioanalyzer
   DNA	Fluorometry
   DNA	Digital PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Time of biospecimen collection	First void Subsequent void Second void Third void Fourth void
   Biospecimen Aliquots and Components	Centrifugation	Different number of centrifugation steps compared Multiple durations compared Multiple speeds compared
   Fluorometry Specific	Technology platform	ddPCR Bioanalyzer
   Storage	Storage duration	2 h 18 h 24 h 48 h 72 h 96 h
   Storage	Storage temperature	Room temperature 4°C 30°C
   Biospecimen Preservation	Type of fixation/preservation	Urine conditioning buffer Cytolyte Streck cfDNA urine presevative

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