Concentration strategies for spiked and naturally present biomarkers in non-invasively collected first-void urine

Abstract

Background: First-void urine (FVU) provides a non-invasive method for collecting a wide range of biomarkers found in genital tract secretions. To optimize biomarker collection in FVU, this study investigated the impact of naturally present and supplemented precipitating agents: uromodulin (UMOD) and polyethylene glycol (PEG), on the concentration of human papillomavirus (HPV) pseudovirions (PsV), cell-free DNA (cfDNA), and cellular genomic DNA (gDNA) through centrifugation.

Methods: FVU samples from ten healthy female volunteers, along with a control sample, were spiked with seal herpesvirus 1 (PhHV-1) DNA, HPV16 plasmid DNA, and HPV16 PsV with an enhanced green fluorescent protein (EGFP) reporter. The samples were subjected to various concentration protocols involving PEG precipitation, low-speed centrifugation (5 min at 1000×g), and medium-speed centrifugation (1 h at 3000×g). Subsequently, quantitative PCR (qPCR) was used to assess cellular and cell-free glyceraldehyde-3-phosphate dehydrogenase (GAPDH) DNA, cell-free PhHV-1 and HPV16 DNA, and PsV (EGFP) DNA. In addition, UMOD levels were measured.

Results: The findings revealed that PEG significantly increased the concentration of cfDNA and gDNA in the pellet after centrifugation, with the most pronounced effect observed for cfDNA. Moreover, low-speed centrifugation without PEG effectively depleted cellular gDNA while preserving cfDNA in the supernatants. Pseudovirions were consistently pelleted, even with low-speed centrifugation, and a positive but not significant effect of PEG on PsV (EGFP) DNA yield in the pellet was observed. Additionally, a significant correlation was observed between UMOD and GAPDH, HPV16, and PsV (EGFP) DNA quantities in the pellet. Furthermore, large variations among the FVU samples were observed.

Conclusions: With this study, we provide novel insights into how various biomarker precipitation protocols, including both the properties of FVU and the use of PEG as a precipitating agent, influence the concentration of cfDNA, cellular gDNA, and pseudovirions.

Keywords: Biomarkers; Concentration; FVU; First-void urine; Human papillomavirus.

Fig. 1

Schematic overview. First-void urine (FVU) samples were collected from 10 female volunteers. Aliquots of 1 ml of the FVU and the control sample were spiked with HPV16 PsV, PhHV-1 DNA, and HPV16 plasmid DNA. Also, one aliquot was not spiked. All the samples underwent biomarker precipitation using six different protocols; (1) precipitation of unspiked samples without PEG (Usp-NP), (2) precipitation of spiked samples without PEG (Sp-NP), (3) precipitation using 10% (w/v) PEG6000, (4) precipitation using 8% (w/v) PEG20000, (5) low-speed centrifugation followed by precipitation of the supernatant using 10% (w/v) PEG6000, and (6) low-speed centrifugation followed by precipitation of the supernatant using 8% (w/v) PEG20000. Supernatants and pellets were collected for all purifications and qPCR was performed for GAPDH, PhHV-1, HPV16, and PsV (EGFP). In addition, ELISA for UMOD was performed

Fig. 2

Boxplots representing the amount of A GAPDH; B PhHV-1; C HPV16; and D PsV (EGFP) DNA in the respective samples. Results are presented as nanograms (ng), arbitrary DNA copies, and DNA copies. The spiked—No PEG arm did not undergo PEG treatment, whereas the other arms were incubated with 10% (w/v) PEG6000 or 8% (w/v) PEG20000 for approximately 16 h at 4 °C. All the samples were centrifuged at 3000×g at 4 °C for 1 h and the low-speed centrifugation (LSC) samples were subjected to low-speed centrifugation without PEG addition at 1000×g for 5 min before subsequent PEG precipitation. Significant differences between the pellet and supernatant for each arm are presented in the figure

Fig. 3

Boxplots representing the amount of A GAPDH; B PhHV-1; C HPV16; and D PsV (EGFP) DNA in the respective samples after various centrifugation steps. Results are presented as ng, arbitrary copies, and DNA copies. All 1 ml samples were spiked with the same amount of PhHV-1 DNA, HPV16 plasmid DNA and HPV16 PsV (EGFP). The samples were centrifuged at 1000×g at 4 °C for 5 min or at 3000×g at 4 °C for 1 h. Centrifugation at 1000×g was performed before both PEG precipitation protocols, resulting in two data points per ID for this arm. Significant differences between the pellet and supernatant for centrifugation conditions are presented in the figure

Fig. 4

Correlation plots between the amount of A GAPDH; B PhHV-1; C HPV16; and D PsV (EGFP) DNA and UMOD in the pellet. Spearman rank correlation coefficients are presented in the figure with, and without (w/o) inclusion of the Sp-NP arm data