Prevalence of and risk factors for curable sexually transmitted infections on Bubaque Island, Guinea Bissau.

OBJECTIVES: Complications from sexually transmitted infections (STIs) can result in severe morbidity and mortality. To date, no STI population studies have been conducted on the Bijagos Islands, Guinea Bissau. Our objective was to estimate the prevalence of and identify risk factors for Chlamydia trachomatis (Ct), Neisseria gonorrhoea (Ng), Mycoplasma genitalium (Mg), Trichomonas vaginalis (Tv) and Treponema pallidum (Tp) on Bubaque, the most populated island. METHODS: A cross-sectional survey was conducted on the island of Bubaque among people aged 16-49 years. Participants were asked to answer a questionnaire on STI risk factors, to provide urine samples (men and women) and vaginal swabs (women) for PCR testing for Ct, Ng, Mg and Tv, and to provide dry blood spots for Tp particle agglutination assays. Data were analysed to estimate the prevalence of STIs and logistic regression was used to identify risk factors. RESULTS: In total, 14.9% of participants were found to have a curable STI, with the highest prevalence being observed for Tv (5.9%) followed by Ct (3.8%), Ng (3.8%), Mg (1.9%) and Tp (0.8%). Significant risk factors for having any STI included being female, younger age and concurrent partnership. Having had a previous STI that was optimally treated was a protective factor. CONCLUSIONS: This study demonstrates that there is a considerable burden of STI on the Bijagos Islands, stressing the need for diagnostic testing to facilitate early detection and treatment of these pathogens to stop ongoing transmission. Moreover, these results indicate the need to conduct further research into the STI burden on the Bijagos Islands to help inform and develop a national STI control strategy.

Development of a multivariable risk model integrating urinary cell DNA methylation and cell-free RNA data for the detection of significant prostate cancer.

BACKGROUND: Prostate cancer exhibits severe clinical heterogeneity and there is a critical need for clinically implementable tools able to precisely and noninvasively identify patients that can either be safely removed from treatment pathways or those requiring further follow up. Our objectives were to develop a multivariable risk prediction model through the integration of clinical, urine-derived cell-free messenger RNA (cf-RNA) and urine cell DNA methylation data capable of noninvasively detecting significant prostate cancer in biopsy naïve patients. METHODS: Post-digital rectal examination urine samples previously analyzed separately for both cellular methylation and cf-RNA expression within the Movember GAP1 urine biomarker cohort were selected for a fully integrated analysis (n = 207). A robust feature selection framework, based on bootstrap resampling and permutation, was utilized to find the optimal combination of clinical and urinary markers in a random forest model, deemed ExoMeth. Out-of-bag predictions from ExoMeth were used for diagnostic evaluation in men with a clinical suspicion of prostate cancer (PSA ≥ 4 ng/mL, adverse digital rectal examination, age, or lower urinary tract symptoms). RESULTS: As ExoMeth risk score (range, 0-1) increased, the likelihood of high-grade disease being detected on biopsy was significantly greater (odds ratio = 2.04 per 0.1 ExoMeth increase, 95% confidence interval [CI]: 1.78-2.35). On an initial TRUS biopsy, ExoMeth accurately predicted the presence of Gleason score ≥3 + 4, area under the receiver-operator characteristic curve (AUC) = 0.89 (95% CI: 0.84-0.93) and was additionally capable of detecting any cancer on biopsy, AUC = 0.91 (95% CI: 0.87-0.95). Application of ExoMeth provided a net benefit over current standards of care and has the potential to reduce unnecessary biopsies by 66% when a risk threshold of 0.25 is accepted. CONCLUSION: Integration of urinary biomarkers across multiple assay methods has greater diagnostic ability than either method in isolation, providing superior predictive ability of biopsy outcomes. ExoMeth represents a more holistic view of urinary biomarkers and has the potential to result in substantial changes to how patients suspected of harboring prostate cancer are diagnosed.

Phenotype Profiling for Forensic Purposes: Determining Donor Sex Based on Fourier Transform Infrared Spectroscopy of Urine Traces.

Forensic science is an important field of analytical chemistry where vibrational spectroscopy, in particular Fourier transform infrared spectroscopy and Raman spectroscopy, present advantages as they have a nondestructive nature, high selectivity, and no need for sample preparation. Herein, we demonstrate a method for determination of donor sex, based on attenuated total reflection Fourier transform infrared (ATR FT-IR) spectroscopy of dry urine traces. Trace body fluid evidence is of special importance to the modern criminal investigation as a source of individualizing DNA evidence. However, individual identification of a urine donor is generally difficult because of the small amount of DNA. Therefore, the development of an innovative method to provide phenotype information about the urine donor-including sex-is highly desirable. In this study, we developed a multivariate discriminant model for the ATR FT-IR spectra of dry urine to identify the donor sex. Rigorous selection of significant wavenumbers on the spectrum using a genetic algorithm enabled superb discrimination performance for the model and conclusively indicated a chemical origin for donor sex differences, which was supported by physiological knowledge. Although further investigations need to be conducted, this proof-of-concept study demonstrates the great potential of the developed methodology for phenotype profiling based on the analysis of urine traces.

Causal somatic mutations in urine DNA from persons with the CLOVES subgroup of the PIK3CA-related overgrowth spectrum.

Congenital lipomatous overgrowth with vascular, epidermal, and skeletal (CLOVES) anomalies and Klippel-Trenaunay (KTS) syndromes are caused by somatic gain-of-function mutations in PIK3CA, encoding a catalytic subunit of phosphoinositide 3-kinase. Affected tissue is needed to find mutations, as mutant alleles are not detectable in blood. Because some patients with CLOVES develop Wilms tumor, we tested urine as a source of DNA for mutation detection. We extracted DNA from the urine of 17 and 24 individuals with CLOVES and KTS, respectively, and screened 5 common PIK3CA mutation hotspots using droplet digital polymerase chain reaction. Six of 17 CLOVES participants (35%) had mutant PIK3CA alleles in urine. Among 8 individuals in whom a mutation had been previously identified in affected tissue, 4 had the same mutant allele in the urine. One study participant with CLOVES had been treated for Wilms tumor. We detected the same PIK3CA mutation in her affected tissue, urine, and tumor, indicating Wilms tumors probably arise from PIK3CA mutant cells in patients with CLOVES. No urine sample from a participant with KTS had detectable PIK3CA mutations. We suggest that urine, which has the advantage of being collected non-invasively, is useful when searching for mutations in individuals with CLOVES syndrome.

The preparation of microfluidic architecture with monolithic materials using a dual porous silica structure.

A microfluidic device (MD) has been developed which features a porous silica (PS) monolithic disk synthesized from tetramethyl orthosilicate, incorporated into the device post-fabrication and sealed in place with a second PS monolithic layer, synthesized from potassium silicate. This dual porous silica (DPS) structure provides a pathway for sample introduction to the MD and offers an ideal platform for solid phase extraction (SPE) methodologies which can be rapidly and efficiently integrated into a chip-based format. All silica disk manufacture and functionalization was carried out in batch to provide a readily scalable method of production. Application of this design for processing samples was demonstrated using two alternative nucleic acid purification chemistries, yielding polymerase chain reaction amplifiable DNA extracted from 150 µL of human urine in less than 35 min. It is proposed that this DPS system could be further developed for a diverse range of chip-based SPE applications, providing an interface facilitating sample delivery and enabling SPE on-chip. Furthermore, to the author's knowledge it is the first reporting of two different types of PS amalgamated in a single MD.

Extraction of cell-free DNA from urine, using polylysine-coated silica particles.

DNA analysis is used for a variety of purposes, including disease diagnosis and DNA profiling; this involves extracting DNA from living organisms. In this study, we prepared polycationic silica particles to extract DNA that has the negatively charged phosphate backbone from solution. The coated particles were prepared by mixing conventional silica gel particles and poly-Lys; these particles could efficiently extract 1.3 µg of cell-free DNA from 50 mL of (male) urine. It is expected that these easily prepared particles (just a mixture of two commercially available chemicals) can be used as a noninvasive diagnostic tool for genetic disorders such as cancer, diabetes, and hypertension. Graphical abstract Effective extraction method of cfDNA from urine was developed that used commercially available silica gel particles and poly-Lys.

A fully validated bioanalytical method using an UHPLC-MS/MS system for quantification of DNA and RNA oxidative stress biomarkers.

A new, rapid and effective ultra-high-performance liquid chromatography method with mass spectrometry detection is described for the separation and quantification of 8-hydroxy-2-deoxyguanosine, 8-hydroxyguanosine and creatinine in human urine. The present study uses an isotope-labelled internal standard ([15N]5-8-hydroxy-2-deoxyguanosine), a BIO core-shell stationary phase and an isocratic elution of methanol and water. Sample preparation of human urine was performed by solid-phase extraction (SPE) on Oasis HLB cartridges with methanol/water 50:50 (v/v) elution. Extraction recoveries ranged from 98.1% to 109.2%. Biological extracts showed high short-term stability. Several aspects of this procedure make it suitable for both clinical and research purposes: a short elution time of less than 3.2 min, an intra-day precision of 2.5-8.9%, an inter-day precision of 3.4-8.7% and low limits of quantification (27.7 nM for 8-hydroxyguanosine, 6.0 nM for 8-hydroxy-2-deoxyguanosine). Finally, simultaneous analysis of DNA and RNA oxidative stress biomarkers is a useful tool for monitoring disease progression in neurodegenerative disorders and cancer. Graphical abstract UHPLC-MS/MS analysis of DNA and RNA oxidative stress biomarkers.

Genetic DNA profile in urine and hair follicles from patients who have undergone allogeneic hematopoietic stem cell transplantation.

Biological samples from patients who have undergone allogeneic hematopoietic stem cell transplantation (HSCT) constitute a challenge for individual identification. In this study we analyzed the genetic profiles (by the amplification of 15 autosomic STRs) of HSCT patients found in different types of samples (blood, hair and urine) that may be the source of DNA in civil or criminal forensic cases. Our results show that while in hair follicles the donor component was not detected in any patient, thus being a reliable source of biological material for forensic identification, mixed chimerism was detected in urine samples from all patient, and no correlation was found between the time elapsed from the transplant and the percentage of chimerism. These results certainly have practical implications if the urine is being considered as a source of DNA for identification purposes in HSTC patients. Moreover, taking into consideration that chimerism was found not only in patients with leukocyturia (given the hematopoietic origin of leukocytes, this was expected), but also in those without observable leukocytes in the sediment, we conclude that an alternative source or sources of donor DNA must be implicated.

[Correlation of genetic and cytogenetic alterations in pathological aggressiveness urothelial carcinoma of the bladder: Performance of BCA-1, a mini-array comparative genomic hybridisation-based test]. / Corrélation des altérations génétiques à l'agressivité anatomo-pathologique des carcinomes urothéliaux de la vessie : performance du test BCA-1.

INTRODUCTION: Urothelial carcinomas are the fourth leading cause of cancer in humans. Their incidence is increasing by more than 50% in 25 years. The superficial forms (70% cases) require a close active surveillance to identify frequent recurrences and progression to invasive stage. Our main goal was to identify prognostic molecular markers for bladder cancer that could be used alone or in combination in routine clinical practice. In this aim, we evaluated the capability of the BCA-oligo test based on a CGH array to correctly classify tumoral grade/stage. METHOD: Urinary DNA was extracted from 81 patients with superficial bladder cancer and has been hybridized on the BCA-oligo array. The results from the molecular analysis were correlated with the tumoral grade and stage. RESULTS: Several chromosomal alterations were significantly more frequent in tumors of higher grade and more advanced stage. A significant association was observed between a high grade and the presence of one of these alterations: loss on 6p, gain on 8q or 13q, loss or gain on 9q or 11q, with an odds ratio of 6.91 (95% CI=2.20-21.64; P=0.0009). Moreover, a significant association was found between a more advanced stage (pT1) and the presence of one of these alterations: loss on 6p, gain on 8q, loss or gain on 5p, with an odds ratio of 15.2 (95% CI=3.71-62.58; P=0.0002). CONCLUSION: Our results showed that molecular analyses of superficial bladder cancers based on urinary DNA and the BCA-oligo test could be used as prognostic factor for the tumor evolution, allowing then a more adapted clinical management.

Exercise-induced oxidatively damaged DNA in humans: evaluation in plasma or urine?

Physical exercise can induce oxidative damage in humans. 8-Hydroxy-2'-deoxyguanosine (8-OHdG) is a widely known biomarker of DNA oxidation, which can be determined in blood and urine. The aim of the present study was to compare these two biological fluids in terms of which is more suitable for the estimation of the oxidative damage of DNA by measuring the concentration of 8-OHdG one hour after maximal exercise by enzyme immunoassay. The concentration of 8-OHdG increased with exercise only in plasma (p < 0.001), and values differed between exercise tests in both plasma and urine (p < 0.05). In conclusion, plasma appears to be more sensitive to exercise-induced 8-OHdG changes than urine and, hence, a more appropriate medium for assessing oxidative damage of DNA, although the poor repeatability of the measurement needs to be addressed in future studies.

Purification of Circulating Cell-Free DNA from Plasma and Urine Using the Automated Large-Volume Extraction on the QIAsymphony® SP Instrument.

Increasing sample numbers for screening and diagnostics using circulating cell-free DNA (ccfDNA) as analyte demands an automated solution for ccfDNA extraction. The efficiency of a new, automated, large volume ccfDNA extraction method was evaluated against a manual reference method. The new kit for automated ccfDNA extraction on the QIAsymphony showed a comparable yield of total ccfDNA from healthy donors as well as a comparable recovery of circulating cancer and fetal DNA. In conclusion, a new kit for automated ccfDNA extraction was established successfully.

Urinary Cell-Free DNA Quantification as Non-Invasive Biomarker in Patients with Bladder Cancer.

INTRODUCTION: Concentration of urinary cell-free DNA (ucfDNA) belongs to potential bladder cancer markers, but the reported results are inconsistent due to the use of various non-standardised methodologies. The aim of the study was to standardise the methodology for ucfDNA quantification as a potential non-invasive tumour biomarker. MATERIAL AND METHODS: In total, 66 patients and 34 controls were enrolled into the study. Volumes of each urine portion (V) were recorded and ucfDNA concentrations (c) were measured using real-time PCR. Total amounts (TA) of ucfDNA were calculated and compared between patients and controls. Diagnostic accuracy of the TA of ucfDNA was determined. RESULTS: The calculation of TA of ucfDNA in the second urine portion was the most appropriate approach to ucfDNA quantification, as there was logarithmic dependence between the volume and the concentration of a urine portion (p = 0.0001). Using this methodology, we were able to discriminate between bladder cancer patients and subjects without bladder tumours (p = 0.0002) with area under the ROC curve of 0.725. Positive and negative predictive value of the test was 90 and 45%, respectively. CONCLUSION: Quantification of ucf DNA according to our modified method could provide a potential non-invasive biomarker for diagnosis of patients with bladder cancer.

Cell-free DNA in the urine of rats exposed to ionizing radiation.

Investigation of cell-free DNA (cf-DNA) in body fluids, as a potential biomarker for assessing the effect of ionizing radiation on the organism, is of considerable interest. We investigated changes in the contents of cell-free mitochondrial DNA (cf-mtDNA) and cell-free nuclear DNA (cf-nDNA) in the urine of X-ray-exposed rats. Assays of cf-mtDNA and cf-nDNA were performed by a real-time PCR in rat urine collected before and after irradiation of animals with doses of 3 and 5 Gy. We also determined the presence of mutations in urine cf-mtDNA, as recognized by Surveyor nuclease. A sharp increase in cf-mtDNA and cf-nDNA in the urine of irradiated rats was observed within 24 h after exposure, followed by a decrease to normal levels. In all cases, the contents of cf-mtDNA fragment copies (estimated by gene tRNA) were significantly higher than those of cf-nDNA estimated by gene GAPDH. A certain portion of mutant cf-mtDNA fragments was detected in the urine of exposed rats, whereas they were absent in the urine of the same animals before irradiation. These preliminary data also suggest that the increased levels of urine cf-mtDNA and cf-nDNA may be a potential biomarker for noninvasive assessment of how the organism responds to ionizing radiation influence.

Molecular Diagnosis of α°-Thalassemia Through Urine DNA: A Novel DNA Source to Facilitate Prevention Programs in Remote Geographical Areas.

We assessed whether urinary DNA sediment was a feasible sample type for the molecular diagnosis of α-thalassemia (α-thal) mutations. Urine samples (5-10 mL) were collected from 218 male and female volunteers. The cells were centrifuged, and DNA was isolated according to the protocol of a commercial DNA isolation kit. Detection of the α(0)-thal [Southeast Asian (- -(SEA)) and - -(THAI)] deletions was performed using quantitative real-time polymerase chain reaction (q-PCR), in addition to conventional gap-PCR. The results revealed that DNA extracted from urinary sediment presented an average DNA content of 11.2 ± 5.5 ng/µL, and the 260/280 ratio indicative of DNA purity, was 1.2 ± 0.2. The overall q-PCR threshold cycle was 31.2 ± 2.3. The melting temperature for the - -(SEA) deletion was 87.3 ± 0.1 °C, while that of the wild type sequence was 92.5 ± 0.2 °C. There were 16 (7.3%) α(0)-thal SEA genotypes detected. These results were in agreement with those of the conventional gap-PCR and blood DNA analyses. Thus, DNA from urinary sediment can be efficiently used for the molecular diagnosis of α(0)-thal mutations. This approach allows for rapid diagnosis, is non invasive, and could be useful for preventing Hb Bart's (γ4) hydrops fetalis syndrome.

[Telomere length and transrenal DNA isolated from transplanted kidney recipients' urine]. / Dlugosc telomerów i transrenalne DNA izolowane z moczu biorców nerki przeszczepionej.

Transplantation is the preferred method of end stage renal insufficiency treatment due to better quality of life and extended life of transplanted patients. Currently a non-invasive test, which evaluates the risk of acute or chronic rejection or deterioration of the transplanted organ's function, is being sought. An increase of the transrenal DNA concentration in the urine of urinary tract infection patients and in renal graft recipients during an episode of acute rejection was observed. There were also reports on shortening of telomeres in transplanted organ chromosomes, as the result of accelerated aging of cells, and its connection with the onset of chronic allograft nephropathy and the degree of its completion, and thus the deterioration of kidney function. The aim of this paper is to describe the urine genetic analysis through determining the length of the telomeres and the content of transrenal DNA to monitor kidney function and to evaluate the prevalence of acute and chronic rejection in patients after kidney transplantation. The genetic analysis of the biological material collected from patients relies on the determination of transrenal DNA content and length of DNA telomeres isolated from the urine of kidney recipients. The presented methods assume that the genetic profile of the transplanted organ recipient as well as kidney donor can be determined, so the source of the genetic material in the urine of the patient can be identified. A measurable effect of these methods' use would be to complement the evaluation of the prevalence of acute and chronic rejection and transplanted kidney function with a modern, non-invasive method, which is the analysis of telomere length from sediment of urine and the content of transrenal DNA in the urine.

Characterization of DNA concentration in urine and dried blood samples to detect the c.577 deletion within the EPO gene.

The EPO gene variant, c.577del (VAR-EPO), was discovered in the Chinese population in 2021. The mutated protein is naturally present in urine from individuals heterozygous for the variant. Electrophoresis methods currently applied in anti-doping laboratories produce a pattern in samples from individuals carrying VAR-EPO that cannot be unambiguously distinguished from individuals who received recombinant EPO doses. Consequently, the analysis of blood samples is obligatory to facilitate interpretation of suspicious findings from urine samples. However, this complicates the process and delays the reporting. Objective of this study was to develop EPO c.577del detection in urine and dried blood samples (DBS) in order to facilitate and accelerate EPO results management. Moreover, estimation of the success rate of sequencing regarding concentration of DNA in urine and DBS was evaluated. Conclusive results regarding Sanger sequencing were obtained for all samples with DNA concentrations above 0.024 ng/µL DNA in 80% of urines samples from volunteers. The potential success of DNA sequencing rate in athletes' urines was investigated. A total of 191 urine samples were considered. DNA concentration exceeding 0.024 ng/µL was detected in 85% of the samples. Interestingly, in-competition samples had a significantly higher DNA concentration than out-of-competition male urine samples (0.330 vs. 0.084 ng/µL). Moreover, conclusive EPO sequences were obtained for 100% of DBS (cellulose and polymer matrices). In conclusion, method for detection of EPO gene variant was developed in urine and DBS. Characterization of DNA concentration was performed in order to evaluate the probability of success of sequencing EPO gene in anti-doping field.

Engineering of a DNA/γPNA Hybrid Nanoreporter for ctDNA Mutation Detection via γPNA Urinalysis.

Detection of circulating tumor DNA (ctDNA) mutations, which are molecular biomarkers present in bodily fluids of cancer patients, can be applied for tumor diagnosis and prognosis monitoring. However, current profiling of ctDNA mutations relies primarily on polymerase chain reaction (PCR) and DNA sequencing and these techniques require preanalytical processing of blood samples, which are time-consuming, expensive, and tedious procedures that increase the risk of sample contamination. To overcome these limitations, here the engineering of a DNA/γPNA (gamma peptide nucleic acid) hybrid nanoreporter is disclosed for ctDNA biosensing via in situ profiling and recording of tumor-specific DNA mutations. The low tolerance of γPNA to single mismatch in base pairing with DNA allows highly selective recognition and recording of ctDNA mutations in peripheral blood. Owing to their remarkable biostability, the detached γPNA strands triggered by mutant ctDNA will be enriched in kidneys and cleared into urine for urinalysis. It is demonstrated that the nanoreporter has high specificity for ctDNA mutation in peripheral blood, and urinalysis of cleared γPNA can provide valuable information for tumor progression and prognosis evaluation. This work demonstrates the potential of the nanoreporter for urinary monitoring of tumor and patient prognosis through in situ biosensing of ctDNA mutations.

Identification of prefoldin amplification (1q23.3-q24.1) in bladder cancer using comparative genomic hybridization (CGH) arrays of urinary DNA.

BACKGROUND: Array-CGH represents a comprehensive tool to discover genomic disease alterations that could potentially be applied to body fluids. In this report, we aimed at applying array-CGH to urinary samples to characterize bladder cancer. METHODS: Urinary DNA from bladder cancer patients and controls were hybridized on 44K oligonucleotide arrays. Validation analyses of identified regions and candidates included fluorescent in situ hybridization (FISH) and immunohistochemistry in an independent set of bladder tumors spotted on custom-made tissue arrays (n = 181). RESULTS: Quality control of array-CGH provided high reproducibility in dilution experiments and when comparing reference pools. The most frequent genomic alterations (minimal recurrent regions) among bladder cancer urinary specimens included gains at 1q and 5p, and losses at 10p and 11p. Supervised hierarchical clustering identified the gain at 1q23.3-q24.1 significantly correlated to stage (p = 0.011), and grade (p = 0.002). The amplification and overexpression of Prefoldin (PFND2), a selected candidate mapping to 1q23.3-q24.1, correlated to increasing stage and tumor grade by means of custom-designed and optimized FISH (p = 0.013 and p = 0.023, respectively), and immunohistochemistry (p ≤0.0005 and p = 0.011, respectively), in an independent set of bladder tumors included in tissue arrays. Moreover, PFND2 overexpression was significantly associated with poor disease-specific survival (p ≤0.0005). PFND2 was amplified and overexpressed in bladder tumors belonging to patients providing urinary specimens where 1q23.3q24.1 amplification was detected by array-CGH. CONCLUSIONS: Genomic profiles of urinary DNA mirrowed bladder tumors. Molecular profiling of urinary DNA using array-CGH contributed to further characterize genomic alterations involved in bladder cancer progression. PFND2 was identified as a tumor stratification and clinical outcome prognostic biomarker for bladder cancer patients.

High-resolution profiling of fetal DNA clearance from maternal plasma by massively parallel sequencing.

BACKGROUND: With the advent of massively parallel sequencing (MPS), DNA analysis can now be performed in a genomewide manner. Recent studies have demonstrated the high precision of MPS for quantifying fetal DNA in maternal plasma. In addition, paired-end sequencing can be used to determine the size of each sequenced DNA fragment. We applied MPS in a high-resolution investigation of the clearance profile of circulating fetal DNA. METHODS: Using paired-end MPS, we analyzed serial samples of maternal plasma collected from 13 women after cesarean delivery. We also studied the transrenal excretion of circulating fetal DNA in 3 of these individuals by analyzing serial urine samples collected after delivery. RESULTS: The clearance of circulating fetal DNA occurred in 2 phases, with different kinetics. The initial rapid phase had a mean half-life of approximately 1 h, whereas the subsequent slow phase had a mean half-life of approximately 13 h. The final disappearance of circulating fetal DNA occurred at about 1 to 2 days postpartum. Although transrenal excretion was involved in the clearance of circulating fetal DNA, it was not the major route. Furthermore, we observed significant changes in the size profiles of circulating maternal DNA after delivery, but we did not observe such changes in circulating fetal DNA. CONCLUSIONS: MPS of maternal plasma and urinary DNA permits high-resolution study of the clearance profile of circulating fetal DNA.

DNA extraction from long-term stored urine.

BACKGROUND: Traditionally, for DNA analyses, DNA is recovered from buffy coats. Since DNA in urine has been reported to deteriorate quickly, this option is often not considered. To complete our DNA database in patients with ANCA-associated vasculitis, we aimed to extract DNA from stored urine. METHODS: Urine was stored at the time of kidney biopsy from patients included in our regional kidney biopsy database, who had given informed consent for further study. Urine was subsequently filtered, dialyzed, concentrated and freeze dried and finally resolubilized and centrifuged. DNA was extracted using the high pure PCR template preparation kit (Roche Diagnostics). Next, concentration and purity were determined by Nanodrop analysis and by Quant-iT analysis. RESULTS: One hundred and eighty-one patients with ANCA-associated vasculitis were included. Of 114 patients (63%), DNA was available. From 53 of the remaining 67 patients, stored urine was available. Of the 53 samples that were processed, 46 (86.8%) yielded DNA with a mean concentration of 258.7 ng/µL (range 33.2-529) with a mean purity ratio of 1.81 (λ 260/280). CONCLUSION: DNA extraction from fresh urine has been described before, yielding DNA usable for PCR analysis in healthy subjects. Storage of fresh urine at 4 °C or lower temperatures results in significant degradation of the DNA, making recovery of DNA more difficult with longer periods of storage. In the current study, we demonstrated that DNA could be retrieved from subsequently filtered, dialyzed, concentrated and freeze dried urine that was stored at room temperature. In addition, we demonstrated that this DNA could be used for PCR analysis. This method is useful when no other material from these patients is available.