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

Abstract

Although liquid biopsies offer many advantages over tissue biopsies, they are not yet standard practice. An important reason for the lack of implementation is the unavailability of well standardized techniques and guidelines, especially for pre-analytical conditions which are an important factor causing the current sensitivity issues. To overcome these limitations, we investigated the effect of several pre-analytical conditions on the concentration of cell-free DNA (cfDNA) and cellular genomic DNA (gDNA) contamination. Urine samples from healthy volunteers (HVs) and cancer patients were collected and processed according to specific pre-analytical conditions. Our results show that in samples with a relatively small volume more than 50% of the cfDNA can be found in the first 50 mL of the urine sample. The total DNA concentration increased again when samples were collected more than 3.5 hours apart. Adding preservative to urine samples is recommended to obtain high concentrations of cfDNA. To remove the cellular content, high speed centrifugation protocols as 4,000g 10min or 3,000g 15min are ideal for urine collected in cfDNA Urine Preserve (Streck). Although this study was a pilot study and needs to be confirmed in a larger study population, clear trends in the effect of several pre-analytical conditions were observed.

Fig 1. Flowchart of all the investigated pre-analytical conditions.

Each condition is shown in a different frame. The number of HVs and patients from which samples were collected and analyzed are displayed on top of each frame. Below the experimental set-up and the used techniques are shown. CP = centrifugation protocol, HVs = healthy volunteers, RT = room temperature, Streck = cfDNA Urine Preserve, UCM = Urine conservation medium.

Fig 2. The percentage of the total DNA, cfDNA and gDNA present in the different fractions of one urine sample derived from five HVs and two patients.

The concentration of total DNA was measured by Qubit Fluorometer and ddPCR. The samples were also analyzed with the FragmentAnalyzer. The percentage of cfDNA and gDNA of each fraction within the entire sample is also visually represented by the grey bars. 100% cfDNA = total amount cfDNA present in the sample according to the measurements of our research team with the mentioned technique. X% cfDNA = X% cfDNA of the 100% cfDNA that is present in the sample based on our measurements. cfDNA = circulating cell-free DNA, ddPCR = digital droplet PCR, FA = FragmentAnalyzer, gDNA = genomic DNA, HV = healthy volunteer, P1/2 = patient 1/2.

Fig 3. First-void versus random urine collection.

Analysis was performed using the FragmentAnalyzer on all included samples. Each dot represents the fraction of a urine sample (in mL) of one HV that is needed to detect 50% of the cfDNA present in this urine sample. In urine samples with a small volume (dots on the left of the vertical black line), the first 50 mL of the urine sample suffices to detect 50% of the present cfDNA in the sample (dots below horizontal black line). In the only urine sample with a large volume that was tested (dots on the right of the vertical black line), the first 50 mL of the urine sample does not suffice to detect 50% of the present cfDNA in the sample (dots above horizontal black line). cfDNA = circulating cell-free DNA, gDNA = genomic DNA, HV = healthy volunteer.

Fig 4. Collection time points.

Different collection points were investigated throughout one entire day starting with the first morning urine with a maximum of five consecutive samples being collected. Results from four HVs are shown as an average for each collection point. A: Total DNA concentrations analyzed with Qubit fluorometer and ddPCR. B: Amount of cfDNA and gDNA as determined with the FragmentAnalyzer. C: The effect on time interval between two consecutive collection points on the total concentration measured with Qubit and ddPCR. cfDNA = circulating cell-free DNA, ddPCR = digital droplet PCR, gDNA = genomic DNA, HV = healthy volunteer.

Fig 5. Effect of preservatives.

The reference fractions were processed immediately after collection. The other fractions were stored for one week at RT with or without the addition of a preservative. Results from all included samples are shown as an average for each fraction. A: Total DNA concentrations analyzed with Qubit fluorometer and ddPCR. B: Amount of cfDNA and gDNA as determined with the FragmentAnalyzer. cfDNA = circulating cell-free DNA, ddPCR = digital droplet PCR, gDNA = genomic DNA, RT = room temperature, Streck = cfDNA Urine Preserve, UCM = Urine conservation medium.

Fig 6. Effect of storage temperature and time in fresh urine samples.

The reference fractions (0h) were processed immediately after collection. The other fractions were stored at RT or 4°C with a varying storage time. Results from all included samples are shown as an average for each fraction A: Total DNA concentrations analyzed with Qubit fluorometer. B: Total DNA concentrations analyzed with ddPCR. C: Amount of cfDNA and gDNA as determined with the FragmentAnalyzer. cfDNA = circulating cell-free DNA, ddPCR = digital droplet PCR, gDNA = genomic DNA, RT = room temperature.

Fig 7. Effect of storage temperature and time in urine samples to which Streck preservative was added.

The fractions were stored at 4°C, RT or 30°C for a varying storage time and supplemented with Streck preservative. Results from all included samples are shown as an average for each fraction. A: Total DNA concentrations analyzed with Qubit fluorometer. B: Total DNA concentrations analyzed with ddPCR. C: Amount of cfDNA and gDNA as determined with the FragmentAnalyzer. cfDNA = circulating cell-free DNA, ddPCR = digital droplet PCR, gDNA = genomic DNA, RT = room temperature, Streck = cfDNA Urine Preserve.

Fig 8. Preparation of cell-free urine by centrifugation.

Five different centrifugation protocols were evaluated using fresh urine samples of HVs. A: Total DNA concentrations analyzed with Qubit fluorometer and ddPCR. B: Amount of cfDNA and gDNA determined with the FragmentAnalyzer. cfDNA = circulating cell-free DNA, CP = centrifugation protocol, CP 1 = 750g 10min + 2,680g 10min, CP 2 = 750g 10min + 3,350g 15min, CP 3 = 750g 20min + 2,680g 10min, CP 4 = 750g 20min, CP 5 = 500g 10min, ddPCR: digital droplet PCR, gDNA = genomic DNA, HV = healthy volunteer.

Fig 9. Evaluation of cfDNA and gDNA concentration for the investigation of centrifugation protocols with preservatives.

Four different centrifugation protocols were evaluated using urine samples with preservative of cancer patients and HVs. A: Total DNA concentrations analyzed with Qubit fluorometer and ddPCR. B: Amount of cfDNA and gDNA determined with the FragmentAnalyzer. cfDNA = circulating cell-free DNA, CP I = 4,000g 10min, CP II = 3,000g 15min, CP III = 2,680g 10min, CP IV = 750g 10min, ddPCR = digital droplet PCR, gDNA = genomic DNA, HV = healthy volunteer.