Sample Collection and Stabilization of Urine and cfDNA

1. Do I need to use PAXgene Urine Liquid Biopsy Tubes only with the PAXgene Urine Collection Cup from the PAXgene Urine Liquid Biopsy Set?
Yes. Further information can be found in the PAXgene Urine Liquid Biopsy Set Handbook accessible under the Resources tab on www.preanalytix.com.

2. Can I use the PAXgene Urine Liquid Biopsy Tube with regular urine collection cup?
The PAXgene Urine Liquid Biopsy Tube is not designed for use with open urine collection cups requiring manual sample transfer into the tube. It is not recommended to manually fill the PAXgene Urine Liquid Biopsy Tube from a syringe or other methods due to increased risk of biohazard exposure, needle stick injury and incorrect urine to additive ratio that may affect product performance. Additionally, removal of the stopper may compromise the sterility of the tube and increase risk of sample contamination.

3. What is the collection volume of the PAXgene Urine Collection Cup?
What is the collection volume of the PAXgene Urine Collection Cup? An individual can void up to 120 mL of urine in the cup. A minimum of 15 mL is recommended to be present in the cup to fill one tube.

4. What is the draw volume of the PAXgene Urine Liquid Biopsy Tube?
What is the draw volume of the PAXgene Urine Liquid Biopsy Tube? The urine draw volume is 10 mL and the additive volume is 1.5 mL. Thus, a filled tube holds a volume of 11.5 mL. Multiple tubes can be drawn from one urine collection cup. Further information can be found in the PAXgene Urine Liquid Biopsy Set Handbook accessible under the Resources tab on www.preanalytix.com.

5. How can I stabilize more than 10 mL from the same urine specimen?
To stabilize more than 10 mL of urine, multiple PAXgene Urine Liquid Biopsy Tubes can be filled from the same cup depending on the total volume of urine collected into the cup. If more than one PAXgene Urine Liquid Biopsy Tube will be filled from the same urine sample, the blue cap of the PAXgene Urine Collection Cup should be unscrewed, slightly lifted and screwed back on without lifting it off completely. Then repeat the steps for each tube as mentioned in the Handbook.

6. I want to stabilize more than 10 mL from the same urine specimen. Can I buy more tubes?
Yes. Further information on ordering additional tubes can be found under Other Products Used in Conjunction on www.preanalytix.com.

7. I followed the instructions on stabilizing, but the tube still filled insufficiently.
To ensure 10 mL of urine is transferred into the PAXgene Urine Liquid Biopsy Tube from the PAXgene Urine Collection Cup, insert the PAXgene Urine Liquid Biopsy Tube with the yellow tube closure facing down into the cavity of the blue cup cap. Push quickly and firmly to puncture the stopper and hold the tube down to begin filling the tube. Hold the PAXgene Urine Liquid Biopsy Tube in place until the tube stops filling. Pushing the tube into the cavity of the blue cup cap slowly may result in tube underfilling. Further, the quantity of urine drawn should be approximately 10 mL for the PAXgene Urine Liquid Biopsy Tube, but this volume may vary depending on various factors such as altitude, ambient temperature, barometric pressure, tube age, and filling technique.

8. Can I stabilize my samples 1 hour after collection into the PAXgene Urine Collection Cup?
The time between urine donation and stabilization of the specimen in the PAXgene Urine Liquid Biopsy Tube is crucial for the successful stabilization of cfDNA. Degradation of cfDNA progresses more rapidly in urine compared to other body fluids like blood if it is not stabilized. Therefore, the time between collection and stabilization should be reduced as much as possible. Ensure the time of specimen collection is recorded and urine is stabilized by transferring into PAXgene Urine Liquid Biopsy Tube within 15 minutes of collection.

9. How does the additive in the PAXgene Urine Liquid Biopsy Tube stabilize cfDNA?
The PAXgene Urine Liquid Biopsy Tube additive is non-crosslinking. Stabilization of cfDNA levels is accomplished by minimizing degradation of cfDNA, minimizing release of gDNA, and minimizing microbial growth during sample storage.

10. What is the additive in the PAXgene Urine Liquid Biopsy Tube?
The PAXgene Urine Liquid Biopsy Tube contains a proprietary formulation that stabilizes cfDNA levels by minimizing degradation of cfDNA, minimizing release of gDNA, and minimizing microbial growth during sample storage. For additional information, visit www.preanalytix.com or review the Safety Data Sheet under the Resources tab.

11. The PAXgene Urine Liquid Biopsy additive has a slightly yellow appearance; does this affect the performance of the additive?
No, a slightly yellow appearance does not affect the performance of the additive as long as the tubes are used before their expiration date.

Storage and Processing

12. What is the shelf life of an unused PAXgene Urine Liquid Biopsy Set?
The shelf life of the set is 12 months from the date of manufacture. The expiration date is printed on the set label.

13. How should an unused PAXgene Urine Liquid Biopsy Set be stored?
Store the unused PAXgene Urine Liquid Biopsy Set at 4–25°C.

14. Is the PAXgene Urine Liquid Biopsy Set sterile?
Yes, the interior of the PAXgene Urine Liquid Biopsy Set components (PAXgene Urine Liquid Biopsy Tube and PAXgene Urine Collection Cup) are sterile. The sterilization method used is gamma irradiation.

15. How long can urine stabilized in the PAXgene Urine Liquid Biopsy Tube be stored at room temperature?
Urine drawn into PAXgene Urine Liquid Biopsy Tube can be stored at 15–25°C for up to 10 days. For further information, please review the Performance Characteristics section of the product handbook.

16. Can urine in the PAXgene Urine Liquid Biopsy Tube be stored at temperatures above 25°C?
Yes. Urine drawn into PAXgene Urine Liquid Biopsy Tubes can be stored for up to 7 days at 30°C or for up to 3 days at 37°C. For further information, please review the Performance Characteristics section of the product handbook.

17. Can urine in the PAXgene Urine Liquid Biopsy Tube be stored at temperatures below 15°C?
Yes. Urine drawn into PAXgene Urine Liquid Biopsy Tube can be stored for up to 10 days at 2–8°C. For further information, please review the Performance Characteristics section of the product handbook.

18. Can urine be frozen in the PAXgene Urine Liquid Biopsy Tube?
No. Freezing of whole urine is not recommended. Cell-free urine supernatant after centrifugation can be transferred into a suitable tube and frozen according to Handbook.

cfDNA Isolation and Processing

19. What is the expected cfDNA yield from 10 mL urine?
The concentration of cfDNA in urine may be low and varies considerably by many factors including among different individuals, sex and health status of the donating individual, time of collection and hydration status.

20. What is the expected cfDNA yield from 2–4 mL urine?
The concentration of cfDNA in urine may be low and varies considerably by many factors including among different individuals, sex and health status of the donating individual, time of collection and hydration status.

21. Which cfDNA isolation kits are compatible with the PAXgene Urine Liquid Biopsy Set?
Isolation of cfDNA from the PAXgene Urine Liquid Biopsy Tube stabilized urine can be performed manually or automated using the QIAGEN QIAamp Circulating Nucleic Acid Kit or the QIAGEN QIAsymphony Circulating DNA Kit (QIAGEN QIAsymphony Circulating DNA Kit is not available in all countries. For further details please contact www.support.qiagen.com)

22. What urine volumes can be processed on the QIAGEN QIAsymphony SP instrument using the QIAGEN QIAsymphony Circulating DNA Kit?
For 10 mL sample input volume, please contact QIAGEN Technical Service. Other sample input volumes are also possible, please visit support.qiagen.com.

23. What urine volumes can be processed using the QIAGEN QIAamp Circulating Nucleic Acid Kit?
For 10 mL sample input volume, see Supplementary Protocols under the Resources tab on www.preanalytix.com. Other sample input volumes are also possible, please visit support.qiagen.com.

24. How should cfDNA isolated from the PAXgene Urine Liquid Biopsy Tube with QIAGEN QIAamp Circulating Nucleic Acid Kit or the QIAGEN QIAsymphony Circulating DNA Kit be stored?
It is recommended to store cfDNA eluates at 2–8°C for up to 1 month or at –20 °C or –80 °C for up to 3 months (long-term study ongoing), or for up to 3 freeze-thaw cycles at –20 °C or –80 °C (tested during storage for 1 month). For further information, please refer to the PAXgene Urine Liquid Biopsy Set Handbook available under resources on www.preanalytix.com

Downstream Applications

25. Which downstream applications have been tested with cfDNA isolated from urine collected and stabilized with the PAXgene Urine Liquid Biopsy Set?
cfDNA isolated from urine collected and stabilized in the PAXgene Urine Liquid Biopsy Set can be used in a range of downstream molecular applications and testing methods requiring cfDNA, such as: PCR, including digital and quantitative real-time PCR.