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

Laura Téblick; Marijana Lipovac; F Ricardo Burdier; Annemie De Smet; Margo Bell; Eef van den Borst; Veerle Matheeussen; Alex Vorsters

doi:10.1186/s40001-024-01719-5

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

Eur J Med Res. 2024 Feb 17;29(1):131. doi: 10.1186/s40001-024-01719-5.

Authors

Laura Téblick¹, Marijana Lipovac², F Ricardo Burdier², Annemie De Smet², Margo Bell², Eef van den Borst^{2

3}, Veerle Matheeussen^{4

5

6}, Alex Vorsters²

Affiliations

¹ Centre for the Evaluation of Vaccination (CEV), Vaccine & Infectious Disease Institute (VAXINFECTIO), Faculty of Medicine and Health Sciences, University of Antwerp, 2610, Wilrijk-Antwerp, Belgium. laura.teblick@uantwerpen.be.
² Centre for the Evaluation of Vaccination (CEV), Vaccine & Infectious Disease Institute (VAXINFECTIO), Faculty of Medicine and Health Sciences, University of Antwerp, 2610, Wilrijk-Antwerp, Belgium.
³ Centre of Medical Genetics, Faculty of Medicine and Health Sciences, University of Antwerp and Antwerp University Hospital, 2650, Edegem, Belgium.
⁴ Department of Microbiology, Antwerp University Hospital (UZA), 2650, Edegem-Antwerp, Belgium.
⁵ Department of Medical Microbiology (LMM), Vaccine & Infectious Disease Institute (VAXINFECTIO), Faculty of Medicine and Health Sciences, University of Antwerp, 2610, Wilrijk-Antwerp, Belgium.
⁶ Department of Medical Biochemistry, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, 2610, Wilrijk-Antwerp, Belgium.

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.

MeSH terms

Biomarkers
Cell-Free Nucleic Acids*
DNA
Female
Human papillomavirus 16* / genetics
Humans

Substances

Cell-Free Nucleic Acids
Biomarkers
DNA

Grants and funding

101040588/ERC_/European Research Council/International