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The Challenge to Stabilize, Extract and Analyze Urinary Cell-Free DNA (ucfDNA) during Clinical Routine.

Author(s): Nel I, Münch C, Shamkeeva S, Heinemann ML, Isermann B, Aktas B

Publication: Diagnostics (Basel), 2023, Vol. 13, Page

PubMed ID: 38132253 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the stability of cfDNA and a synthetic cfDNA reference standard in pooled urine specimens that were preserved with Urine Analyte Stabilizer (UAS), Urine Conditioning Buffer (UCB), or a homemade buffer (AlloU) and then stored at room temperature or 4°C for up to 24 h. The authors also compared the yield of cfDNA from urine specimens collected from male and female volunteers. Urine was collected from four healthy male volunteers and four healthy female volunteers. Urine aliquots from each individual were frozen at -80°C and the remaining urine was pooled. The pooled urine was aliquoted and the cfDNA reference standard that contained the EGFR T790M mutation was added to half of the aliquots. Matched aliquots of pooled urine were left unpreserved or preserved with UAS, UCB, or the AlloU buffer (EDTA, diazolidinyl urea, imidazolidinyl urea, and Tris-HCl pH 8) and stored at room temperature or 4°C for 0, 4, 12, and 24 h. At each timepoint, the urine was centrifuged at 3000 g for 10 min, and the supernatant was frozen at -80°C.  Urine aliquots were thawed for 1 h at room temperature, and the cell debris was removed by centrifugation at 16,000 g. cfDNA was extracted from pooled urine and individual volunteer urine aliquots using the QIAamp MinElute ccfDNA Mini Kit with a 25 µL extraction volume. To test the effect of elution volume, cfDNA was isolated from pooled urine that was preserved with UAS and stored at room temperature for 0 and 2 h; cfDNA yield was compared among samples with an elution volume of 20 and 50 µL. cfDNA was quantified using the Qubit dsDNA HS Assay Kit. Wildtype and T790M-mutated EGFR were detected by droplet digital PCR in cfDNA extracted from UCB- and AlloU-preserved urine from the 0, 4, and 24 h timepoints.

   Summary of Findings:

   Although more cfDNA was obtained when an elution volume of 50 µL was used rather than 20 µL (26.25 vs. 19.19 ng), an elution volume of 20 µL resulted in a more consistent yield. cfDNA yield varied widely between individuals, with < 3 ng cfDNA obtained from 5 of 8 urine specimens and 73.53 ng cfDNA obtained from one volunteer. The mean cfDNA yield was non-significantly higher in urine specimens from females than males (19.75±31.44 ng versus 5.37±5.85 ng, P=0.15), but this was largely due to the high yield of cfDNA from one volunteer. In the unpreserved urine without the reference standard, cfDNA yield was stable for 12 h at 4°C and then increased between 12 h and 24 h (P=0.022 compared to 12 h) but was significantly higher after 4 h than 0 h (P=0.044) and then declined between 12 h and 24 h (P=0.036) when stored at room temperature.  Storage of non-preserved urine with the reference DNA showed similar trends to the urine without reference DNA when specimens were stored at room temperature, but cfDNA yield increased between 0 and 4 h (P=0.008) and again between 12 and 24 h (P=0.022) when stored at 4°C. cfDNA levels changed over the timecourse for all preservatives evaluated at both storage temperatures, but significance was dependent on the preservative, storage temperature, and the addition or omission of the reference standard DNA. Preservation with UAS or AlloU allowed for greater recovery of the reference standard DNA compared to unpreserved urine or urine that was preserved with UCB; the greatest recovery from urine stored for 12 h at room temperature occurred when specimens were preserved with UAS, and the greatest recovery from urine stored for 12 h at 4°C occurred when specimens were preserved with AlloU.  cfDNA levels (without reference standard) were higher in unpreserved or UAS-preserved urine than AlloU- or UCB-preserved urine. However, only small changes in cfDNA levels were observed over the storage timecourse when urine was preserved with AlloU or UCB and stored at 4°C or preserved with UCB and stored at room temperature. While the fractional abundance (FA) of EGFR T790M was relatively stable over the 24 h timecourse in urine specimens that were preserved with UAS, the FA was lower than expected at approximately 2% (1.9-3.4%) rather than the expected 5%. The authors stated that this can be accounted for by the relatively high abundance of cfDNA in the urine specimen. In AlloU-preserved urine, the FA was also lower than expected (2.25-2.95%), even when accounting for the cfDNA in the urine, until 12 h at either room temperature or 4°C; EGFR T790M was not detectable after storage for 24 h at room temperature.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • Other Preservative
   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Digital PCR
   DNA	Fluorometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Patient gender	Female Male
   Storage	Storage duration	0 h 4 h 12 h 24 h
   Storage	Storage temperature	4°C Room temperature
   Biospecimen Aliquots and Components	Biospecimen components	Reference standard DNA added No reference standard DNA added
   Biospecimen Preservation	Type of fixation/preservation	UAS Urine conditioning buffer AlloU None (fresh)

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