ORCID Profile
0000-0002-9515-3020
Current Organisation
Garvan Institute of Medical Research
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Publisher: Springer Science and Business Media LLC
Date: 19-06-2017
DOI: 10.1007/S40291-017-0284-X
Abstract: Blood s les for studies of circulating DNA in disease are often collected in clinical settings where prompt processing of s les is not possible. In order to avoid problems associated with leukocyte lysis after prolonged blood storage, stabilised blood tubes have been developed containing preservatives that prevent cell lysis. We evaluated Streck BCT tubes and PAXgene ccfDNA tubes, as well as standard EDTA blood collection tubes, in terms of DNA yield and fragment size. Blood was collected in EDTA, Streck BCT or PAXgene ccfDNA tubes and stored for 1 h at 4 °C, or 4 days at room temperature. DNA was extracted using the QIA Circulating Nucleic Acids kit, and visualised on an agarose gel or quantitated by qPCR. Ratios of a 247-base and a 115-base licon of the Alu repetitive element were used to infer size distribution. While plasma DNA in EDTA tube blood s les increased by ~10- to 20-fold after 4 days of storage at room temperature, both Streck BCT tubes and PAXgene ccfDNA tubes maintained stable plasma DNA concentrations. A slight decrease in DNA yield following 1 h of blood storage at 4 °C was observed in Streck BCT and PAXgene ccfDNA tubes relative to EDTA tubes. This decrease was reversed by increasing the proteinase digest step of the DNA extraction protocol to 60 min, as recommended by Streck tube product literature. Visualisation of the extracted DNA on an agarose gel showed that after 4 days of room temperature storage, s les collected in EDTA tubes contained abundant high-molecular weight DNA, which was partially fragmented in a ladder pattern. A slight increase in high-molecular weight DNA in s les stored for 4 days at room temperature in Streck BCT tubes was also observed, but this was not reflected in a change in large and small Alu fragment ratios as measured by qPCR. Tubes containing preservative to prevent cell lysis can extend the scope for blood collection in clinical settings however, slight differences between s les collected in different tube types underscore the requirement for standardised protocols, as well as attention to s le handling.
Publisher: Public Library of Science (PLoS)
Date: 26-04-2021
DOI: 10.1371/JOURNAL.PONE.0250561
Abstract: Assays measuring cell-free DNA (cfDNA) in blood have widespread potential in modern medicine. However, a comprehensive understanding of cfDNA dynamics in healthy in iduals is required to assist in the design of assays that maximise the signal driven by pathological changes, while excluding fluctuations that are part of healthy physiological processes. The menstrual cycle involves major remodelling of endometrial tissue and associated apoptosis, yet there has been little investigation of the impact of the menstrual cycle on cfDNA levels. Paired plasma s les were collected from 40 healthy women on menstruating (M) and non-menstruating (NM) days of their cycle. We measured total cfDNA by targeting ALU repetitive sequences and measured endothelial-derived cfDNA by methylation-specific qPCR targeting an endothelium-unique unmethylated CDH5 DNA region. CfDNA integrity and endothelial cfDNA concentration, but not total cfDNA, are consistent across time between NM and M. No significant changes in total (ALU-115 p = 0.273 ALU-247 p = 0.385) or endothelial cell specific (p = 0.301) cfDNA were observed, leading to the conclusion that menstrual status at the time of diagnostic blood collection should not have a significant impact on the quantitation of total cfDNA and methylation-based cancer assays.
Publisher: Walter de Gruyter GmbH
Date: 31-05-2022
Abstract: Circulating DNA (cirDNA) is generally purified from plasma that has been biobanked for variable lengths of time. In long-term experiments or clinical trials, the plasma can be stored frozen for up to several years. Therefore, it is crucial to determine the stability of cirDNA to ensure confidence in s le quality upon analysis. Our main objective was to determine the effect of storage for up to 2 years on cirDNA yield and fragmentation. We stored frozen EDTA plasma and purified cirDNA from 10 healthy female donors, then quantified cirDNA yield at baseline, and at regular intervals for up to 2 years, by qPCR and Qubit. We also compared cirDNA levels in non-haemolysed and haemolysed blood s les after 16 months of storage and tested the effect of varying DNA extraction protocol parameters. Storage up to two years caused an annual cirDNA yield decline of 25.5% when stored as plasma and 23% when stored as purified DNA, with short fragments lost more rapidly than long fragments. Additionally, cirDNA yield was impacted by plasma input and cirDNA elution volumes, but not by haemolysis. The design of long-term cirDNA-based studies and clinical trials should factor in the deterioration of cirDNA during storage.
Publisher: Elsevier BV
Date: 03-2022
DOI: 10.1016/J.RBMO.2021.11.006
Abstract: Do women with laparoscopically confirmed endometriosis have higher plasma concentrations of circulating cell-free DNA (cirDNA) than those without endometriosis? Prospective study of women aged 18-45 years undergoing benign gynaecological laparoscopy at two tertiary hospitals. Venous blood was collected immediately before surgery, and women were allocated to the endometriosis or control groups based on surgical findings. Total plasma cirDNA and cirDNA integrity were measured by quantitative polymerase chain reaction (qPCR) targeting short (115 bases) and long (247 bases) ALU segments. Endometrial-derived cirDNA was measured by qPCR of bisulfite-treated cirDNA using primers selective for a FAM101A sequence uniquely unmethylated in endometrial tissue. Five cirDNA parameters were compared between the control and endometriosis cohorts: total cirDNA concentration, long-stranded cirDNA concentration, integrity ratio, endometrial cirDNA concentration and endometrial cirDNA proportion. Twenty-eight endometriosis and 15 control s les were included. Women with and without endometriosis had cirDNA concentrations of 2.24 ± 0.89 ng/ml and 2.56 ± 0.92 ng/ml, respectively. Analysis by phenotype of endometriosis revealed a significantly higher endometrial cirDNA concentration in women with superficial disease (n = 10) compared with deep endometriosis (n = 18) (mean difference 0.14 ng/ml 95% CI 0.15 to 0.26 P = 0.025), but not with controls. No significant differences were found in any of the cirDNA parameters between women with and without endometriosis. The low statistical power and heterogenous pelvic pathology in the control group render it difficult to determine whether the negative results reflect a true lack of increase in cirDNA in endometriosis.
Publisher: Elsevier BV
Date: 12-2018
DOI: 10.1016/J.CANCERGEN.2018.02.004
Abstract: The utility of circulating DNA as a source of clinical biomarkers in blood is limited by its low concentration and small fragment size. Effective purification methods can maximize circulating DNA yield and contribute to the success of downstream protocols. We describe the evaluation of 4 commercial DNA purification kits-QIA Circulating Nucleic Acids kit, QIA DNA Blood Mini kit, QIA Ultrasens Virus kit and the QIASymphony DSP Virus kit-for the extraction of high and low molecular weight DNA from blood plasma. Using qPCR to quantitate endogenous Alu sequences, as well as spiked exogenous high and low molecular weight zebrafish DNA, we found that the Circulating Nucleic Acids kit and the DSP kit were both efficient at purifying DNA from plasma regardless of fragment size, whereas the DNA Blood Mini kit was only able to effectively extract high molecular weight DNA. The Ultrasens Virus Kit produced the lowest yields for both large and small fragments. The use of carrier RNA with the Circulating Nucleic Acids and the DSP kits improved yields. Appropriate choice of kit can be an important factor in determining experiment outcome.
Publisher: Public Library of Science (PLoS)
Date: 25-10-2019
Publisher: eLife Sciences Publications, Ltd
Date: 09-11-2021
DOI: 10.7554/ELIFE.69679
Abstract: Research and clinical use of circulating cell-free DNA (cirDNA) is expanding rapidly however, there remain large gaps in our understanding of the influence of lifestyle and biological factors on the amount of cirDNA present in blood. Here, we review 66 in idual studies of cirDNA levels and lifestyle and biological factors, including exercise (acute and chronic), alcohol consumption, occupational hazard exposure, smoking, body mass index, menstruation, hypertension, circadian rhythm, stress, biological sex and age. Despite technical and methodological inconsistences across studies, we identify acute exercise as a significant influence on cirDNA levels. Given the large increase in cirDNA induced by acute exercise, we recommend that controlling for physical activity prior to blood collection is routinely incorporated into study design when total cirDNA levels are of interest. We also highlight appropriate selection and complete reporting of laboratory protocols as important for improving the reproducibility cirDNA studies and ability to critically evaluate the results.
Publisher: Oxford University Press (OUP)
Date: 27-09-2022
Abstract: Is circulating cell-free DNA (cirDNA) from the endometrium elevated during menstruation and in endometriosis? Endometrial cirDNA does not increase during menstruation and is not elevated in endometriosis. Changes in cirDNA associated with common benign conditions are a potential source of false positives in cancer diagnostic applications, but also present an opportunity for biomarker development for diseases such as endometriosis. Elevated cirDNA has been reported in endometriosis patients compared to healthy community controls, but no difference in total or endometrial cirDNA has been found between patients with endometriosis and patients with other gynaecological conditions. Likewise, menstruation is a potential driver of changes in cirDNA levels and tissue profile, but total and endothelial cirDNA do not increase during menstruation. For endometriosis comparisons, 59 participants with surgically confirmed endometriosis and 27 laparoscopic patients without endometriosis (hospital controls) were prospectively recruited, while 25 healthy community participants (healthy controls) were recruited in a university setting. Total and endometrial cirDNA and cirDNA fragmentation were measured across the three groups. For menstrual comparisons, 36 matched non-menstruating and menstruating s les were collected from healthy women recruited within a university setting, and the endometrial cirDNA was compared between the two groups. cirDNA was extracted from venous blood plasma then quantitated by quantitative PCR of ALU repetitive element (115 bp) and TP53 gene sequence (105 bp) for total concentration. cirDNA derived from the endometrium was quantitated by methylation-specific droplet digital PCR of a FAM101A region (69 bp) after bisulfite conversion of the DNA. A cirDNA size fragmentation ratio was obtained by quantifying a long segment of ALU repetitive element (247 bp) and expressing the amount relative to the 115 bp ALU target. No differences in cirDNA level were found in any comparison populations in this study. Mean total cirDNA was unchanged between healthy controls (ALU-115–3.31 ng/ml TP53–2.73 ng/ml), hospital controls (ALU-115–3.47 ng/ml TP53–2.83 ng/ml) and endometriosis patients (ALU-115–3.35 ng/ml TP53–2.66 ng/ml). Likewise, endometrial cirDNA was unchanged between healthy controls (18.3 copies/ml), hospital controls (20.6 copies/ml) and endometriosis patients (22 copies/ml). Endometrial cirDNA did not change during menstruation (non-menstruating: 38 copies/ml menstruating: 33 copies/ml). Irrespective of endometriosis diagnosis, blood from patients undergoing laparoscopy (hospital controls: 0.77 endometriosis patients: 0.79), had a significantly higher cirDNA size ratio than community-recruited healthy controls (0.64), indicating increased abundance of long cirDNA fragments. It was not possible to completely match the age, BMI and parity between the three cohorts investigated, however of these, only age has been shown to influence circulating DNA levels and not within the age range of our cohort. Blood from community-recruited healthy women and women undergoing laparoscopy was collected via antecubital vein venepuncture (processed within 3 h) and with either peripheral cannula or venepuncture (processed within 6 h), respectively, which could potentially impact the size distribution of circulating DNA fragments. For the collection of non-menstruating phase blood s les, we did not differentiate between follicular phase, ovulation and luteal phase. Thus, only the mensturating s les were collected at a consistent phase, and any fluctuations in cirDNA that occur at the other phases may have obscured small changes during menstruation. There is no evidence that cirDNA has potential as a diagnostic biomarker for endometriosis. Endometriosis, representing a common benign gynaecological condition, and menstruation, representing a normal physiological occurrence in women, should not affect methylation-based diagnostics in other disease areas, including oncology. N.L.Y.: Australian Government Research Training Program (RTP) Stipend through The University of New South Wales, Translational Cancer Research Network PhD Scholarship Top-Up Award via the Cancer Institute NSW, Beth Yarrow Memorial Award in Medical Science, UNSW Completion Scholarship C.E.H.: Gynaecological Oncology Fund of the Royal Hospital for Women K.W.: Ovarian Cancer Research Foundation and CAMILLA AND MARC. C.E.F.: UNSW Women’s Wellbeing Academy and the Australian Human Rights Institute. We declare the following competing interest: K.W. holds stock in Guardant Health, Exact Sciences and Epigenomics AG. No other authors have competing interests. N/A.
No related grants have been discovered for Nicole Laurencia Yuwono.