Storage Of Urine Samples Research Articles

The utility of an HPV genotype assay for cancer screening in self-collected urine/vaginal samples from Japanese women.

Objectives The high incidence of invasive cervical cancer among those who have not undergone cancer screening is a serious problem. This study aimed to investigate the utility of HPV test results from self-collected urine and vaginal samples as screening tools. Design The study was conducted in two steps. First, the appropriate storage container, temperature, and time until urine HPV assay performance were verified. Second, the results of spot urine testing under those conditions and of gynecologist-collected cervical and self-collected vaginal samples were compared to verify the feasibility of using the BD Onclarity® HPV assay for individuals with abnormal cervical cytology. Participants/Materials, Setting, Methods The participants were 121 women with abnormal cervical cytology. Self-collected urine and vaginal samples, along with gynecologist-collected cervical samples, were tested for HPV using the BD Onclarity® HPV assay. The optimal conditions for urine sample storage were identified by comparing the HPV detection rates under various conditions. Results Urine stored in a BD Probe Tec™ (QxUPT) for less than 72 hours at room temperature was found to have the highest HPV positivity rate. Under these conditions, the detection rates of HPV in urine, cervical, and vaginal samples were examined. HPV type 16 was detected in 41.7% of the cervical samples, type 18 in 10%, and types 31 and 52 in 12.6% each. The concordance rate for HPV testing between clinician-collected cervical and urine samples was 63.9% (kappa 0.34; 95% CI: 0.21-0.47), and that between clinician-collected cervical and self-collected vaginal samples was 77.8% (kappa 0.68; 95% CI: 0.53-0.83), indicating good concordance. In a population with an HPV-related lesion/tumor prevalence of approximately 70%, the sensitivity of HPV testing was 82.7% for the cervix, 46.4% for urine, and 75.7% for vaginal samples. Limitations The primary limitation is the lower detection rate of HPV in spot urine samples than in other sample types, indicating room for methodological improvement. The study's findings are based on a specific population, which may limit generalizability. Conclusions We investigated the optimal self-collected urine-to-testing time and temperature. Self-collected vaginal and urine HPV tests show moderate-high concordance with clinician-collected cervical HPV tests, suggesting their potential utility for women who do not undergo regular cancer screening. However, the sensitivity was not high in spot urine. Therefore, further large-scale studies are needed to verify these findings and optimize testing methods to encourage broader participation in cancer screening programs.

Validation of a LC-MS/MS assay for citric acid, cysteine and oxalic acid determination and its application to explore pre-analytical sample storage

ObjectivesCitrate, oxalate and cystine in 24-h urine are considered to be associated with the incidence and recurrence risk of urinary stone disease (USD). An evaluation of the LC-MS/MS kit for simultaneous quantification of the three analytes was undertaken. Design& Methods: The analytical performance of the kit was investigated based on FDA, EMA and CLSI guidelines. To promote the standardization of sample storage, this kit has been applied to perform systematic pre-analytical stability study of these analytes in urine. ResultsThis method was validated with good linearity with accuracy of 93.1%–104 %. Intra-day and inter-day imprecision were ≤5.55 % and 5.34 %, respectively. Recoveries of citrate, oxalate and cystine added to clinical samples were in the range of 92.0–103 %, 94.8–100 % and 99.0–107 % with CV ≤ 5.52 %. It was recommended that urine preserved with hydrochloric acid could be preferable in consideration of both reliable test results and neglected sample heterogeneity. ConclusionsThis kit is suitable for measurement of citrate, oxalate and cystine for understanding the etiology of urinary stones, and the proper storage of urine samples is crucial for the correctness of the test results.

Detection of boldenone in the urine of female horses-ex vivo formation versus administration.

Boldenone is an anabolic-androgenic steroid (AAS) that is prohibited in equine sports. However, in certain situations, it is endogenous, potentially formed by the microbes in urine. An approach to the differentiation based on the detection of the biomarkers Δ1-progesterone, 20(S)-hydroxy-Δ1-progesterone and 20(S)-hydroxyprogesterone was assessed, and their concentrations were monitored in the urine of untreated female horses (n = 291) alongside boldenone, boldienone, testosterone and androstenedione. Using an ultra-sensitive analytical method, boldenone (256 ± 236 pg/mL, n = 290) and the biomarkers (Δ1-progesterone up to 57.6pg/mL, n = 8; 20(S)-hydroxy-Δ1-progesterone 85.3 ± 181 pg/mL, n = 130; 20(S)-hydroxyprogesterone 43.5 ± 92.1pg/mL, n = 158) were detected at low concentrations. The ex vivo production of Δ1-steroids was artificially induced following the storage of urine samples at room temperature for 7days in order to assess the concentrations and ratios of the monitored steroids. The administration of inappropriately stored feed source also resulted in an increase in 20(S)-hydroxy-Δ1-progesterone concentrations and the biomarker ratios. Using the results from different datasets, an approach to differentiation was developed. In situations where the presence of boldenone exceeds a proposed action limit of 5ng/mL, the presence of the biomarkers would be investigated. If Δ1-progesterone is above 50 pg/mL or if 20(S)-hydroxy-Δ1-progesterone is above 100 pg/mL with the ratio of 20(S)-hydroxy-Δ1-progesterone:20(S)-hydroxyprogesterone greater than 5:1, then this would indicate ex vivo transformation or consumption of altered feed rather than steroid administration. There remains a (small) possibility of a false negative result, but the model increases confidence that adverse analytical findings reported in female horses are caused by AAS administrations.

Development and validation of a UPLC-MS/MS method for the quantification of sugars and non-nutritive sweeteners in human urine

High-intensity sweeteners (‘sweeteners’), such as sucralose, saccharine, acesulfame, cyclamate and steviol, are replacing sugars in many food products, but biomarker-based data on their population-wide exposure, as well as analytical methods that can quantify urinary concentrations of sugars and sweeteners simultaneously, are lacking. Here, we developed and validated an ultra-pressure liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) method to quantify glucose, sucrose, fructose, sucralose, saccharine, acesulfame, cyclamate and steviol glucuronide in human urine. Urine samples were prepared by a simple dilution step containing the internal standards in water and methanol. Separation was achieved on a Shodex Asahipak NH2P-40 hydrophilic interaction liquid chromatography (HILIC) column using gradient elution. The analytes were detected using electrospray ionization in negative ion mode, and selective reaction monitoring was optimized using the [M−H]- ions. Calibration curves ranged between 34 and 19,230 ng/mL for glucose and fructose, and 1.8 to 1,026 ng/mL for sucrose and the sweeteners. The method has acceptable accuracy and precision, which depends on the application of appropriate internal standards. Storage of urine samples in lithium monophosphate gives the best overall analytical performance, and storage at room temperature without any preservatives should be avoided since this leads to reduced glucose and fructose concentrations. With the exception of fructose, all analytes were stable throughout 3 freeze–thaw cycles. The validated method was applied to human urine samples, demonstrating quantifiable concentrations of the analytes which were in the expected range. It is concluded that the method has acceptable performance to quantitatively determine dietary sugars and sweeteners in human urine.

The impact ofurine storage methods onthe results ofquantitative bacterial culture indog and cat.

Quantitative bacterial culture (QBC) isthe gold standard for determining urinary tract infections. However, the majority ofurine samples were sent toalab for further bacterial culture. Therefore, the storage condition was vital tomaintain the quality and accuracy ofthe urine samples. The main objective ofour study was toexamine the urine QBC outcomes of(1) immediate culture, (2) culture after urine sample storage atambient temperature for 24 h, and (3) culture after urine sample storage at4°C for 24hours. There were 49 feline samples and 30 canine samples included inthis study. All QBC samples kept atambient temperature and refrigerator were consistent with immediate QBC incats. Eight positive results from immediate QBC were inaccordance with refrigerated results indogs. There were ten positive results inthe room-temperature sample with two false-positive results. Our study showed that storing conditions atroom temperature orrefrigeration for 24 h does not impact the results ofQBC incat urine samples. For dog samples, chilled samples have ahigher accuracy rate than room temperature samples, although the overall agreement was still satisfactory.

Effects of Variation in Urine Sample Storage Conditions on 16S Urogenital Microbiome Analyses.

Replicability is a well-established challenge in microbiome research with a variety of contributing factors at all stages, from sample collection to code execution. Here, we focus on voided urine sample storage conditions for urogenital microbiome analysis. Using urine samples collected from 10 adult females, we investigated the microbiome preservation efficacy of AssayAssure Genelock (Genelock), compared with no preservative, under different temperature conditions. We varied temperature over 48 h in order to examine the impact of conditions samples may experience with home voided urine collection and shipping to a central biorepository. The following common lab and shipping conditions were investigated: -20°C, ambient temperature, 4°C, freeze-thaw cycle, and heat cycle. At 48 h, all samples were stored at -80°C until processing. After generating 16S rRNA gene amplicon sequencing data using the highly sensitive KatharoSeq protocol, we observed individual variation in both alpha and beta diversity metrics below interhuman differences, corroborating reports of individual microbiome variability in other specimen types. While there was no significant difference in beta diversity when comparing Genelock versus no preservative, we did observe a higher concordance with Genelock samples shipped at colder temperatures (-20°C and 4°C) when compared with the samples shipped at -20°C without preservative. Our results indicate that Genelock does not introduce a significant amount of microbial bias when used on a range of temperatures and is most effective at colder temperatures. IMPORTANCE The urogenital microbiome is an understudied yet important human microbiome niche. Research has been stimulated by the relatively recent discovery that urine is not sterile; urinary tract microbes have been linked to health problems, including urinary infections, incontinence, and cancer. The quality of life and economic impact of UTIs and urgency incontinence alone are enormous, with $3.5 billion and $82.6 billion, respectively, spent in the United States. annually. Given the low biomass of urine, novelty of the field, and limited reproducibility evidence, it is critical to study urine sample storage conditions to optimize scientific rigor. Efficient and reliable preservation methods inform methods for home self-sample collection and shipping, increasing the potential use in larger-scale studies. Here, we examined both buffer and temperature variation effects on 16S rRNA gene amplicon sequencing results from urogenital samples, providing data on the consequences of common storage methods on urogenital microbiome results.

High-Coverage Quantitative Metabolomics of Human Urine: Effects of Freeze-Thaw Cycles on the Urine Metabolome and Biomarker Discovery.

Urine sample storage after collection at ultra-low-temperature (e.g., -80 °C) is normally required for comparative metabolome analysis of many samples, and therefore, freeze-thaw cycles (FTCs) are unavoidable. However, the reported effects of FTCs on the urine metabolome are controversial. Moreover, there is no report on the study of how urine FTCs affect biomarker discovery. Herein, we present our study of the FTC effects on the urine metabolome and biomarker discovery using a high-coverage quantitative metabolomics platform. Our study involved two centers located in Hangzhou, China, and Edmonton, Canada, to perform metabolome analysis of two separate cohorts of urine samples. The same workflow of sample preparation and dansylation isotope labeling LC-MS was used for in-depth analysis of the amine/phenol submetabolome. The analysis of 320 samples from the Hangzhou cohort consisting of 80 healthy subjects with each urine being subjected to four FTCs resulted in relative quantification of 3682 metabolites with 3307 identified or mass-matched. The analysis of 176 samples from the Edmonton cohort of 44 subjects with four FTCs quantified 3516 metabolites with 3166 identified or mass-matched. Multivariate and univariate analyses indicated that significant variations (fold change ≥ 1.5 with q-value ≤ 0.05) from FTCs were only observed in a very small fraction of the metabolites (<0.3%). Moreover, various metabolites did not show a consistent pattern of concentration changes from one to four FTCs, allowing the use of two separate cohorts of samples to remove these randomly changed metabolites. Three metabolite biomarkers for separating males and females were discovered, and FTC did not influence their discovery.

Kidney-Derived Methylated PAX2 Sequences in the Urine of Healthy Subjects as a Convenient Model for Optimizing Methylation-Based Liquid biopsy.

Although urine-based liquid biopsy has received considerable attention, there is a lack of a simple model to optimize assay parameters, including cell-free DNA (cfDNA) extraction, bisulfite modification, and bis-DNA recovery after conversion for methylation analysis in urine. The primary aim of this work was to establish a practical model by developing a quantitative methylation-sensitive PCR (qMS-PCR) assay for PAX2 based on hypermethylated PAX2 cfDNA that couldbedetected in healthy human urine. We firststudiedthe methylation status of PAX2 in kidney tissues and whole blood,followedbyanassessment ofcommercial kits for bisulfite conversion and bis-DNA recovery. Furthermore, we investigated the influence of urine storage and collection conditions on the preservation of methylated PAX2 in urine samples by qMS-PCR. As expected, PAX2 methylation was identified in urine but not in blood. Two commercial kits (CellCook and Zymo Research) had similar conversion efficiency and bis-DNA recovery. Urine storage for up to 5days did not changePAX2methylation estimates. Overall, cold storage of urine samples and the CellCook urine container maintained higher levels of methylated PAX2 compared to urine kept at room temperature and the conventional tubes, respectively. These findings highlight the importance of using the correct approaches/kits and optimizing experimental conditions as a diagnostic tool in the clinical setting. Our study provides insights on the development of urine-based liquid biopsy with DNA methylation as a universal biomarker.

Differentiation of boldenone administration from ex vivo transformation in the urine of castrated male horses.

Boldenone is an anabolic-androgenic steroid that is prohibited in equine sports. However, in certain situations, it is endogenous or is believed to be formed by microbes in urine, and therefore, an approach for the differentiation is required. Following the identification of Δ1-progesterone and 20(S)-hydroxy-Δ1-progesterone as potential biomarkers of microbial activity, the presence of six steroids was investigated in the postrace urine of castrated male horses (geldings, n = 158). In line with endogenous findings from several other species when ultrasensitive methods are employed, boldenone was detected at low concentrations in all urine samples (27.0-1330 pg/ml). Furthermore, testosterone and androstenedione were detected in 157 samples (≤12,400 and 944 pg/ml, respectively), boldienone in two samples (≤22.0pg/ml) and 20(S)-hydroxy-Δ1-progesterone in 20 samples (≤66.0pg/ml). Δ1-Progesterone was not detected in any population samples analysed on arrival at the laboratory. The ex vivo transformation of boldienone, boldenone, androstenedione, Δ1-progesterone and 20(S)-hydroxy-Δ1-progesterone was induced following the storage of urine samples at room temperature for 7days but not after refrigeration. Because the administration of inappropriately stored feed sources also resulted in an increase in 20(S)-hydroxy-Δ1-progesterone concentrations, a biomarker approach to distinguish steroid administrations was proposed. In situations where the presence of boldenone would exceed a proposed action limit, the presence of Δ1-progesterone and 20(S)-hydroxy-Δ1-progesterone would be investigated. If either Δ1-progesterone or 20(S)-hydroxy-Δ1-progesterone would exceed 50 and 100 pg/ml, respectively, for instance, then this would indicate ex vivo transformation or consumption of altered feed rather than steroid administration.

Non-invasive assessment of metabolic responses to food restriction using urinary triiodothyronine and cortisol measurement in macaques

Regulation of energy allocation and metabolic rate plays an important role in determining behavior and fitness in wild animals, calling for the validation of non-invasive markers of energetic condition. Recently, the thyroid hormone triiodothyronine (T3) has emerged as a promising marker as concentrations decrease to lower the metabolic rate during energetically challenging periods. However, it remains largely unclear whether T3 merely represents an alternative or provides additional information compared to other compounds involved in the regulation of energy acquisition and allocation, like cortisol and C-peptide, as few joint measurements have been conducted to date in non-invasively collected samples. We aimed to validate the non-invasive measurement of immunoreactive urinary total T3 (uTT3), in comparison to urinary cortisol (uCort) and urinary C-peptide (uCP), as a marker of metabolic response to variation in food intake in macaques, and to address a number of issues regarding the collection, storage and processing of samples which are important for application of uTT3 measurements under field conditions. We used daily samples and body mass measures from a prior food restriction-refeeding experiment over 4 weeks with six captive macaques and analyzed concentrations of uTT3 and uCort in samples collected prior to (fasting) and after morning feeding (non-fasting). Concentrations of uTT3 decreased in response to restriction in food supply and were also lower during weeks of food restriction compared to weeks of refeeding. Variation in uTT3 also correlated positively with variation in body mass and concentrations of uCP. As expected, uCort showed the reverse pattern, increasing during food restriction and decreasing following refeeding, but was not associated with variation in body mass. Generally, compared to fasting samples, concentrations were higher in post-morning feeding, i.e. non-fasting, samples for uTT3 but not uCort. Contamination of urine samples with fecal matter, but not soil, and exposure to UV light led to a decrease in uTT3. uTT3 was largely unaffected by repeated freeze–thaw cycles and by refrigeration for medium-term storage (2 days) but degraded substantially when stored at ambient temperature for the same period. In conclusion, uTT3 measurements inform on the effect of food intake and its associated metabolic response to variation in energetic status. Since uTT3 is reasonably robust to many issues associated with collection and storage of urine samples under field conditions, it is a promising biomarker for studies of energetic condition and basal metabolic rate in wild macaques.

Pre-analytical and analytical variables that influence urinary volatile organic compound measurements.

There has been rapidly accelerating interest in the utilization of volatile organic compounds (VOCs) as non-invasive methods for rapid point-of-care medical diagnostics. There is widespread variation in analytical methods and protocols, with little understanding of the effects of sample storage on VOC profiles. This study aimed to determine the effects on VOC profiles of different storage times, at room temperature, prior to freezing, of sealed urine samples from healthy individuals. Analysis using Field Asymmetric Ion Motility Spectrometry (FAIMS) determined the alterations in VOC and total ion count profiles as a result of increasing room temperature storage times. Results indicated that increasing exposure time to room temperature prior to freezing had a threefold effect. Firstly, increased urinary VOC profile variability, with a plateau phase between 12 and 48 hours, before further degradation. Secondly, an increase in total ion count with time exposed to room temperature. Finally, a deterioration in VOCs with each sample run during the analysis process. This provides new insight into the effect of storage of urine samples for VOC analysis using FAIMS technology. Results of this study provide a recommendation for a 12-hour maximum duration at room temperature prior to storage.

Bladder Cancer Cell Capture: Elucidating the Effect of Sample Storage Conditions on Capturing Bladder Cancer Cells via Surface Immobilized EpCAM Antibody.

The gold standard to detect bladder cancer, cystoscopy, is an invasive procedure requiring ambulant hospitalization, thus presenting an obstacle for routine diagnosis. We aim to develop a noninvasive detection method as an alternative that selectively captures shed cancer cells in the patient's urine via surface-immobilized anti-EpCAM antibody. However, the urine sample storage conditions prior to analysis affect the subsequent cancer cell capture rates by the device. In this study, we investigate the capture rates of HT1197 and HT1376 bladder cancer cells in different media (fresh and aged urine as well as PBS) and storage temperatures prior to analysis (37 and 4 °C) as well as in the presence of adjuvants in the medias (free antibodies and cell debris). Capture efficiencies decreased in as little as 1 h of the sample being incubated at 37 °C in all media studied here. Furthermore, cell debris played a strong part in reducing the capture efficiency. From the data, we conclude that storing the sample at 4 °C resulted in the best capture efficiency if storage of more than 1 h was required, which gave valuable insights for this sensor's translation from laboratory to real-world applications.

Levels of tin and organotin compounds in human urine samples from Iowa, United States

Exposure to tin in the general US population is near ubiquitous, as determined using urinary tin levels measured by inductively coupled plasma mass spectrometry (ICP-MS). Urinary tin levels are associated with chronic health outcomes, such as diabetes; however, it is unclear if these associations are due to the presence of inorganic and organic forms of tin in urine. To address this knowledge gap, levels of total tin and several organotin compounds (OTCs) were measured in convenience urine samples from pregnant women and adults from Iowa, United States. Total tin and OTC levels in urine samples were quantified using ICP-MS and gas chromatography with pulsed flame photometric detection (GC-PFPD), respectively. ICP-MS detected tin in almost all urine samples from both study populations. Low levels of dibutyltin were detected in two out of fifty human urine samples. Importantly, storage of urine samples in plastic containers, but not HNO3-pretreated glass vials drastically reduced the recoveries of OTCs, in particular, tributyltin. Although their detection frequency is low, exposures to OTC should be considered when studying associations between human exposures to tin compounds and adverse health outcomes; however, urinary OTC levels measured in banked urine samples may not be suitable as biomarkers of OTC exposure.

The Real-World Frequency of 24-Hour Urine Protein Electrophoresis (UPEP), Serum Free Light Chain (SFLC), and Serum Protein Electrophoresis (SPEP) Testing in Patients with Multiple Myeloma (MM)

IntroductionUp to 20% of MM patients present with light-chain-only multiple myeloma (LCMM), which is not detectable using serum protein electrophoresis testing. 24-hour urinalysis may be the guideline-endorsed method for monitoring LCMM, but impracticalities can limit its use. We sought to assess how patients with MM and LCMM were followed for progression in the community setting by specimen type using real-world data, analyzing the frequency of testing for patients with MM (based on SPEP) or LCMM (based on UPEP or SFLC).MethodsThis retrospective cohort study used information from the Flatiron Health database, which is derived from electronic health record data; Institutional Review Board approval with a waiver of informed consent was obtained. The cohort included 4591 patients followed for MM and LCMM from 01/01/2011 to 06/30/2018 at community oncology clinics across the United States. The cohort was limited to patients with a confirmed diagnosis of MM on or after 01/01/2011, who received at least one line of therapy and did not receive a stem cell transplant. Patients whose start of MM treatment (captured through chart review) was > 30 days before the start of structured activity in the database were excluded as this may indicate missing therapy data. The specimen type was the test used to follow patients for progression throughout their disease course. Tests were eligible to define the specimen type if they occurred ≤ 90 days before or any time after an initial MM diagnosis. If a patient had multiple test types within this timeframe, the specimen type was determined using a hierarchy (at least one detectable SPEP [≥ 1 g/dL] > at least one detectable 24-hour UPEP [≥ 200 mg] > SFLC [any numeric value]), consistent with International Myeloma Working Group (IMWG) criteria. A LCMM diagnosis was assumed when a detectable M spike (≥1 g/dL) via SPEP could not be found 90 days before or any time after the initial MM diagnosis date, but a detectable lab value was found within that timeframe via 24-hour UPEP (≥ 200mg) or a SFLC (any numeric value).ResultsAmong all patients in the cohort with MM (N=4591), 27.7% (N=1272) received a UPEP test, 58.1% (N=2666) received a SFLC test, and 79.2% (N=3635) received a SPEP test at baseline, defined as occurring within 90 days on either side of the MM diagnosis date. Patients with a UPEP specimen type had less frequent testing than patients with a SPEP or SFLC specimen type. For patients with a UPEP specimen type (N=229), the median of the median time between two tests was 95 days, compared to 42 days for patients with a SFLC specimen type (N=1274) and 35 days for patients with a SPEP specimen type (N=2536). 32.7% (N=1503) of the cohort had LCMM; of those patients, 84.8% were followed by SFLC while only 15.2% were followed by UPEP.Conclusions24-hour urine tests for UPEP are not used often in routine clinical practice despite IMWG guidelines: the guidelines may not reflect a realistic standard of care in the real-world setting. While performing UPEP in addition to SFLC regularly may be the ideal practice for monitoring LCMM, impracticalities such as issues with collection technique and proper storage of urine samples may be hindering its regular use in the real world. The SFLC test may present a more realistic alternative for evaluation of disease status in patients with LCMM, and its ease of use could facilitate more frequent serial testing. This in turn could improve the measurement of progression at more regular intervals, potentially reducing patient exposure to therapies that are no longer effective. To our knowledge, this is the first study to investigate the differences in utilization of UPEP, SFLC, and SPEP testing conducted to follow MM and LCMM progression in the real world. [Display omitted] DisclosuresFoster:Flatiron Health: Employment. Lipitz:Flatiron Health: Employment. Torres:Flatiron Health: Employment. Carson:Roche: Consultancy; Flatiron Health: Employment; Washington University in St. Louis: Employment.

Urine metabolome analysis by gas chromatography–mass spectrometry (GC–MS): Standardization and optimization of protocols for urea removal and short-term sample storage

BackgroundBefore derivatization, urine analyzed by gas chromatography–mass spectrometry (GC–MS) requires the complete removal of urea to avoid interferences. We aimed at establishing the most effective sample pretreatment for urea removing; moreover, we explored the impact of two short-term sample storage conditions on urine metabolome. Methods92 aliquots were obtained from a single sample collected from a healthy adult; they were divided into 6 groups. Group 1 consisted of untreated aliquots while groups 2–6 differed from each other for the addition of various defined urease solution volumes combined with either 30 min or 1-hour sonication time. Urine sample storage was tested by comparing 20 fresh aliquots analyzed after collection with 20 aliquots frozen at −80 °C for 72 h. Resultsthe most effective protocol consisted of the combination between 200 μL urease solution with 1-h sonication time; urease solution volumes >200 μL increase the risk to underestimate metabolite peaks because of sample dilution. Short-term storage of samples at −80 °C pointed out significant changes in the urine metabolic profile compared with that of fresh samples. Conclusionsour study confirms the importance of urea removal for a reliable recognition and quantitation of metabolites; urine short-term storage at −80 °C should be carefully reconsidered.

Aldosterone breakthrough in dogs with naturally occurring myxomatous mitral valve disease

IntroductionAldosterone breakthrough (ABT) is the condition in which angiotensin converting enzyme inhibitors (ACEIs) and/or angiotensin receptor blockers fail to effectively suppress the activity of the renin angiotensin aldosterone system. The objective of this study was to determine if ABT occurs in dogs with naturally occurring myxomatous mitral valve disease receiving an ACEI, using the urine aldosterone to creatinine ratio (UAldo:C) as a measure of renin angiotensin aldosterone system activation. Animals, Materials and MethodsThis study includes 39 dogs with myxomatous mitral valve disease. A UAldo:C cut-off definition (derived from a normal population of healthy, adult, and client-owned dogs) was used to determine the prevalence of ABT in this population. Spearman analysis and univariate logistic regression were used to evaluate the relationship between UAldo:C and ABT (yes/no) and eight variables (age, serum K+ concentration, serum creatinine concentration, ACEI therapy duration and ACEI dosage, furosemide therapy duration and furosemide dosage, and urine sample storage time). Finally, the UAldo:C in dogs receiving spironolactone, as part congestive heart failure (CHF) therapy, was compared to dogs with CHF that were not receiving spironolactone. ResultsThe prevalence of ABT was 32% in dogs with CHF and 30% in dogs without CHF. There was no relationship between either the UAldo:C or the likelihood of ABT and the eight variables. Therapy with spironolactone lead to a significant elevation of the UAldo:C. DiscussionUsing the UAldo:C and a relatively stringent definition of ABT, it appears that incomplete RAAS blockade is common in dogs with MMVD receiving an ACEI. The prevalence of ABT in this canine population mirrors that reported in humans. While the mechanism of ABT is likely multifactorial and still poorly understood, the proven existence of ABT in dogs offers the potential to improve the prognosis for MMVD with the addition of a mineralocorticoid receptor blocker to current therapeutic regimens. ConclusionsApproximately 30% of dogs being treated for heart disease and CHF satisfied the definition of ABT. Identifying patient subpopulations experiencing ABT may help guide future study design and clinical decision-making.

Abstract 424: Urine cf-DNA SNPs as early biomarkers of prostate cancer

Abstract Background: The current strategies of prostate cancer (PC) detection, such as serum PSA, digital rectal examination (DRE) and prostate biopsy have been hampered by many limitations, including the degree of aggressiveness, invasiveness, non-specificity to PCa, over diagnosis and over treatment. Recent studies have proposed the use of cell-free circulating DNA (cf-DNA) as non-invasive biomarkers for cancer diagnosis, prediction of tumor burden and prognosis. We hypothesize that a selective subset of PC associated Single Nucleotide Polymorphisms (SNPs) in urine cf-DNA detects PCa with high specificity and sensitivity. Aim: To identify PC specific SNPs in urine cf-DNA from prostate cancer (PC) patients and further validate their clinical utilities as non-invasive biomarkers for early detection of PC in comparison to other genitourinary conditions, such as benign prostate hyperplasia (BPH) and prostatitis. Method and Results: A protocol was optimized for collection, processing and storage of urine samples and isolation of urine cf-DNA. Our discovery dataset was constructed by comparative Illumina HiSeq next generation sequencing (NGS) of urine cf-DNA from three different cohorts of patients presenting with PC (Gleason score 6), benign prostatic hyperplasia (BPH) or prostatitis. The data was analyzed by SNAPE-pooled and annotated using ANNOVAR; revealing 24 PC-specific non-synonymous SNPs. The selective detection of urine cf-DNAs in PC patients was verified by conventional PCR using primers flanking their genomic sites. Our validation analysis was performed in patients (n = 50/group) diagnosed with PC (Gleason score ≤ 3+4), Gleason score ≥ than 4 + 3, or patients deemed as “no cancer seen”. This was performed by Droplet Digital PCR (ddPCR) targeting wild-type versus variant (SNP) DNA in each patient sample. The results have revealed detection of two PC specific SNPs in urine cf-DNA (&amp;gt;60% for SNP1 and &amp;gt;75% SNP2 in 91 patients; patent pending). Conclusion: Our study brings forth the potential clinical utility for two novel urine cf-DNA SNPs as early biomarkers for prostate cancer. Citation Format: Amrita Datta, Hogyoung Kim, Allison Feibus, Melody Badoo, Adedoyin Johnson, Ahmed Moustafa, Jonathan Silberstein, Krishnarao Moparty, Raju Thomas, Asim B. Abdel-Mageed. Urine cf-DNA SNPs as early biomarkers of prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 424.

A method to monitor urinary carbon dioxide in patients with septic shock

Severe sepsis and septic shock are life-threatening conditions with mortality rates exceeding 31% (Levy et al., 2012) [1]. Septicemia was the most expensive US hospital condition in 2011 (Torio and Andrews, 2006) [2]. Urinary carbon dioxide may provide rapid, clinically useful information about a patient’s status, empowering physicians to intervene earlier and improve septic shock mortality. The objective of this paper is to validate a protocol with a Severinghaus-type CO2 probe for the measurement of urinary CO2 of septic shock patients. This protocol includes (i) sampling urine from a Foley catheter in an intensive care unit setting, (ii) storing samples until analysis at a separate facility, (iii) calibration of the Severinghaus-type CO2 sensor, and (iv) measuring urinary CO2 levels. We discuss the preparation and stability of standard solutions, storage of urine samples, and the performance characteristics of the Severinghaus type CO2 sensor in relation to mock urine samples, urine quality control standards, and urine samples from healthy volunteers as well as patients in severe sepsis or septic shock. We report the influence of the sample pH, temperature, and ionic strength on the precision and accuracy of the measurements of urinary CO2 levels and show that the protocol developed for the quantitative assessment of urinary CO2 levels is adequate for the analysis of urine samples collected from a Foley catheter.

Processing of voided urine for prostate cancer RNA biomarker analysis.

Voided urine samples have been shown to contain cells released from prostate tumors. Could good quality RNA from cells in urine be obtained from every donor for multimarker analysis? In addition, could urine donation be as simple as possible, a practical consideration for a lab test, without involving a prostate massage (as indicated for PCA3 testing), which precludes frequent collection; needing it done at a specific time of day (e.g., first or second urine); and requiring prompt processing of samples in clinics with limited molecular biology capability? Collected urine samples were pelleted, and the RNA isolated was processed for cDNA synthesis and in vitro transcription to generate amplified sense aRNA. The resultant aRNA was rigorously analyzed for possible introduced changes. DMSO was used as a cell preservative for frozen storage of urine samples. Good quality aRNA was obtained for over 100 samples collected at two different institutions. The process of RNA amplification removed co-isolated DNA in some samples, which did not affect RNA amplification. Amplification did not amplify genes that were absent and produce other expression alterations. The sense aRNA could be used to generate urinary transcriptomes specific to individual patients. No chaotropic agents for RNA preservation were added to the urine samples so that the supernatant could be used for analysis of secreted protein biomarkers. The time of donation was not important since patients were seen during the entire day. DMSO was an effective cell preservative for freezing urine. Urinary RNA can be readily isolated and amplified for prostate cancer biomarker analysis. Individual patients had unique set of transcripts derived from their tumor.

Long-term Stability of Urinary Biomarkers of Acute Kidney Injury in Children

Recent meta-analyses support the utility of urinary biomarkers for the diagnosis and prognosis of acute kidney injury. It is critical to establish optimal sample handling conditions for short-term processing and long-term urinary storage prior to widespread clinical deployment and meaningful use in prospective clinical trials. Prospective study. 80 children (median age, 1.1 [IQR, 0.5-4.2] years) undergoing cardiac surgery with cardiopulmonary bypass at our center. 50% of patients had acute kidney injury (defined as≥50% increase inserum creatinine from baseline). We tested the effect on biomarker concentrations of short-term urine storage in ambient, refrigerator, and freezer conditions. We also tested the effects of multiple freeze-thaw cycles, as well as prolonged storage for 5 years. Urine concentrations of neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule 1 (KIM-1), and interleukin 18 (IL-18). All biomarkers were measured using commercially available kits. All 3 biomarkers were stable in urine stored at 4°C for 24 hours, but showed significant degradation (5.6%-10.1% from baseline) when stored at 25°C. All 3 biomarkers showed only a small although significant decrease in concentration (0.77%-2.9% from baseline) after 3 freeze-thaw cycles. Similarly, all 3 biomarkers displayed only a small but significant decrease in concentration (0.84%-3.2%) after storage for 5 years. Only the 3 most widely studied biomarkers were tested. Protease inhibitors were not evaluated. Short-term storage of urine samples for measurement of NGAL, KIM-1, and IL-18 may be performed at 4°C for up to 24 hours, but not at room temperature. These urinary biomarkers are stable at-80°C for up to 5 years of storage. Our results are reassuring for the deployment of these assays as biomarkers in clinical practice, as well as in prospective clinical studies requiring long-term urine storage.