Quantification of circulating cell-free DNA (cfDNA) in urine using a newborn piglet model of asphyxia

Abstract

Cell free DNA (cfDNA) in plasma has been described as a potential diagnostic indicator for a variety of clinical conditions, including neonatal hypoxia. Neonatal hypoxia or perinatal asphyxia is a severe medical condition caused by a temporary interruption in oxygen availability during birth. Previously, we have reported temporal changes of cfDNA detected in blood in a newborn piglet model of perinatal asphyxia. However, cfDNA can also be found in other body liquids, opening for a less invasive diagnostic prospective. The objective of this study was to test and establish a reliable method for the isolation and quantification of cfDNA from urine and to explore changes in the quantities of cfDNA using a newborn piglet model of asphyxia. Animals were exposed to hypoxia-reoxygenation (n = 6), hypoxia-reoxygenation + hypothermia (n = 6) or were part of the sham-operated control group (n = 6) and urine samples (n = 18) were collected at 570 minutes post-intervention. Two alternative applications of cfDNA measurement were tested, an indirect method comprising a centrifugation step together with DNA extraction with magnetic beads versus a direct assessment based on two centrifugation steps. CfDNA concentrations were determined by a fluorescent assay using PicoGreen and by qRT-PCR. Genomic (gDNA) and mitochondrial DNA (mtDNA) cfDNA were determined in parallel, taking into account potential differences in the rates of damages caused by oxidative stress. In contrast to previous publications, our results indicate that the direct method is insufficient. Application of the indirect method obtained with the fluorescence assay revealed mean cfDNA levels (SD) of 1.23 (1.76) ng/ml for the hypoxia samples, 4.47 (6.15) ng/ml for the samples exposed to hypoxia + hypothermia and 2.75 (3.62) ng/ml for the control animals. The mean cfDNA levels in piglets exposed to hypoxia + hypothermia revealed significantly higher cfDNA amounts compared to mean cfDNA levels in the samples purely exposed to hypoxia (p < 0.05); however, no significant difference could be determined when compared to the control group (p = 0.09). Application of the indirect method by qRT-PCR revealed mean cfDNA levels of mtDNA and gDNA at the detection limit of the technique and thus no reliable statistics could be performed between the observed cfDNA levels in the investigated groups. The methodology for detection and monitoring of cfDNA in urine has to be further optimized before it can be applied in a clinical setting in the future.

Fig 1. Direct and indirect method for cfDNA quantification.

Schematic presentation of the two methods tested for cell free DNA (cfDNA) assessment in urine samples. (A.) A direct method with simply centrifugation and (B.) an indirect method with cfDNA enrichment based on cfDNA extraction using magnetic beads. For cfDNA quantification, all samples were measured by a fluorescent assay and by qRT-PCR, with primers detecting genomic (gDNA) and mitochondrial DNA (mtDNA).

Fig 2. The newborn piglet model of asphyxia exposed to hypoxia-reoxygenation.

Newborn piglets were randomized into three study arms: hypoxia-reoxygenation, hypoxia-reoxygenation + hypothermia or sham-operated controls. Perinatal asphyxia was mimicked by breathing 8% O₂ and CO₂ aiming at a PaCO₂ of 8.0–9.5 kPa until a BE of -20 mmol/l and/or mean blood pressure fell below 20 mm Hg. Urine samples were collected at post-intervention, 570 minutes after the end of hypoxia. They were obtained using suprapubic aspiration, transferred to 2 ml tubes and frozen at -80°C.

Fig 3. CfDNA concentrations, measured in urine samples of newborn piglets exposed to hypoxia-reoxygenation, processed according to different methods.

For quantification of cfDNA in the urine samples, collected using a newborn piglet model of asphyxia with the interventions strategies hypoxia-reoxygenation ("hypoxia"), hypoxia-reoxygenation + hypothermia ("hypothermia") or sham-operated controls ("control"), the following strategies were tested: (A.) CfDNA levels in samples processed according to the direct method. No statistically significant difference between the groups was found. (B.) CfDNA levels in samples measured using the indirect method. When measured with fluorescence, cfDNA concentrations in the hypoxia + hypothermia group were higher compared to cfDNA levels in the hypoxia group, but not when compared to controls (p = 0.09). QRT-PCR investigating mtDNA and gDNA targets, revealed higher cfDNA levels in the hypoxia + hypothermia group when compared to the controls and to the hypoxia group; however, cfDNA values were at the detection limit of the technique.