Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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UAS™-A Urine Preservative for Oncology Applications.

Author(s): Jordaens S, Arora A, MacDonald KW, Wood C, Hendrickx JO, Zwaenepoel K, Deben C, Tjalma W, Pauwels P, Beyers K, Vankerckhoven V

Publication: Cancers (Basel), 2023, Vol. 15, Page

PubMed ID: 37370729 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the stability of human, microbial, and host-cell cfDNA in unpreserved urine and urine preserved with UAS after storage of urine for 7 or 14 days at room temperature and after 0 or 3 freeze-thaw cycles. To investigate the effects of delayed processing, first-morning urine was collected from 7 volunteers (five females and 2 males; diagnosis not specified) into standard urine collection cups or Colli-Pee® FV-5020 containers. Urine specimens were aliquoted and transported on ice packs to the laboratory where they were centrifuged immediately or after 7 days at room temperature (20-26°C); supernatants were frozen at -80°C until cfDNA extraction. To evaluate if UAS can preserve cfDNA in urine, first morning urine was collected from 42 males and 42 females into Colli-Pee® FV-5020 containers. Fourteen pooled specimens from females and males were created by pooling urine specimens from three patients. Each urine pools were split and UAS preservative was added to half while the other half remained unpreserved. The preserved and unpreserved urine was then aliquoted and stored at room temperature for 0, 7, or 14 days or subjected to 0 or 3 freeze-thaw cycles (-20°C to 40°C; ≥ 3 h at each temperature) before centrifugation. After relevant storage, urine aliquots were centrifuged at 3800 x g for 20 min, filtered through a 0.8 µm filter, and stored at -80°C until cfDNA extraction. Urine was thawed at 37°C, and cfDNA was extracted from the supernatant using the QIAamp Circulating Nucleic Acid Kit and from the pellet (UAS experiments only) using the PowerFecal Pro DNA Kit. cfDNA was quantified by real-time PCR amplification of 16S Bacterial rDNA, TS143, and β-globin. DNA fragment size was profiled using a High-Sensitivity D5000 DNA ScreenTape machine.

    

   Summary of Findings:

   Compared to urine aliquots processed immediately, urine stored for 7 days at room temperature had significantly less β-globin (human) cfDNA (average CT 6.58 cycles higher, P<0.0001) and more microbial (16S) cfDNA (average CT 2.83 cycles lower, P=0.0367). When stored for 7 days at room temperature, the change in CT relative to unstored specimens for microbial, host-cell (TS143) and β-globin cfDNA were much higher in unpreserved urine than urine preserved with UAS (-7.64 versus 0.30 cycles, P=0.0002; 6.05 versus 0.07 cycles, P<0.0001; and 8.25 versus 0.18 cycles, P<0.0001, respectively).  The change in CT for TS143 and β-globin cfDNA in specimens stored for 14 days was also higher in unpreserved urine than urine preserved with UAS (9.61 versus -0.01 cycles, P<0.0001; and 7.68 versus 0.33 cycles, P<0.0001, respectively). UAS preserved urine also showed significantly smaller changes in average CT for β-globin and TS143 cfDNA after 3 freeze-thaw cycles than unpreserved urine (0.32 versus 7.49 cycles, P<0.0001; and 0.31 versus 6.68 cycles, respectively P<0.0001).  Interestingly, delayed processing did not affect the cfDNA fragment size profile.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • None (Fresh)
   • Other Preservative

   Diagnoses:

   • Normal
   • Not specified

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR
   DNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Time at room temperature	0 days 7 days 14 days
   Biospecimen Preservation	Type of fixation/preservation	UAS None (fresh)
   Storage	Freeze/thaw cycling	0 cycles 3 cycles
   Real-time qPCR Specific	Targeted nucleic acid	Human cfDNA (β-globin) Host cell cfDNA (TS143) Microbial DNA (16S rDNA)
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated

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2. Study Purpose

   This study compared the concentration and percentage of cfDNA extracted using three different kits from UAS preserved urine of male and female volunteers, cancer patients, and pregnant women. Midday first-void urine samples were collected from four healthy female volunteers, six healthy male volunteers, seventeen pregnant women, five breast cancer patients, and five prostate cancer patients into Colli-Pee UAS FV-5040 containers. Urine was immediately centrifuged at 3000 x g for 10 min and the supernatant was aliquoted. cfDNA was extracted from matched aliquots using the Norgen Urine Cell-Free Circulating DNA Purification Kit, the Promega Maxwell RSC Circulating DNA Purification Kit, and the Qiagen QIAamp Circulating Nucleic Acid (CNA) Kit. cfDNA was quantified using the Qubit dsDNA High Sensitivity Assay Kit, and the DNA profile and cfDNA percentage were determined using the Cell-Free DNA ScreenTape Assay.

   Summary of Findings:

   The concentration of cfDNA isolated from the three extraction kits ranged from 0.00-236.53 ng/mL. The concentration of cfDNA was higher in urine from female than male healthy volunteers (98.43±58.05 ng/mL versus 2.30±2.33 ng/mL) and in urine from pregnant women or women with breast cancer than men with prostate cancer (33.28±19.02 ng/mL and 31.04±15.87 ng/mL, respectively, versus 4.63±3.66 ng/mL), but in all groups the percentage cfDNA was approximately 20%. Extraction of cfDNA with the Norgen urine Kit resulted in significantly lower cfDNA concentrations than when extraction was with the MaxWell RSC (P<0.03) or QIAamp CNA Kit (P<0.0001), but extraction method did not affect the percentage cfDNA observed.

   Biospecimens

   • Bodily Fluid - Urine

   Preservative Types

   • Other Preservative

   Diagnoses:

   • Neoplastic - Germ Cell
   • Pregnant
   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry
   DNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	Norgen Urine Cell-Free Circulating DNA Purification Kit Promega Maxwell RSC Circulating DNA Purification Kit Qiagen QIAamp Circulating Nucleic Acid Kit
   Preaquisition	Patient gender	Female Male

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