Storage Of Urine Samples Research Articles

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.

Effect of freeze/thaw cycles on several biomarkers in urine from patients with kidney disease.

Urine samples were collected from eleven randomly selected patients with kidney disease, including diabetic nephropathy, chronic nephritis, and nephritic syndrome. Urine samples were treated with one of four protocols for freezing and thawing: freeze directly and thaw directly; freeze directly and thaw by temperature gradient; freeze by temperature gradient and thaw directly; and freeze by temperature gradient and thaw by temperature gradient. After one to six freeze/thaw cycles at -20°C or -80°C, different biomarkers showed differential stabilities. The concentrations of total protein, calcium, and potassium did not change significantly after five freeze/thaw cycles at either -20°C or -80°C. Albumin could only sustain three freeze/thaw cycles at -20°C before it started to degrade. We recommend that urine be stored at -80°C as albumin and the organic ions could sustain five and six freeze/thaw cycles, respectively, using the simple "direct freeze and direct thaw" protocol. Furthermore, in most cases, gradient freeze/thaw cycles are not necessary for urine sample storage.

Urine storage under refrigeration preserves the sample in chemical, cellularity and bacteriuria analysis of ACS

INTRODUCTION: The analysis of urine abnormal constituents and sediment (ACS) comprises tests of great diagnostic and prognostic value in clinical practice. When the analysis of ACS cannot be performed within two hours after collection, the sample must be preserved in order to avoid pre-analytical interferences. Refrigeration is the most applied technique due to its cost effectiveness. Moreover, it presents fewer inconveniences when compared to chemical preservation. However, changes in ACS may also occur in samples under refrigeration. OBJECTIVE: To analyze the influence of refrigeration at 2 to 8ºC on the storage of urine samples within 24 hours. MATERIAL AND METHOD: A total of 80 urine samples were selected from patients admitted at Universidade Federal de Juiz de Fora (UFJF) university hospital, which were tested for ACS at room temperature and stored under refrigeration for 6, 12 and 24 hours. RESULTS: The results showed that refrigeration proved to be effective when compared to samples kept at room temperature, inasmuch as the physical, chemical, microbial and cellularity features were preserved. Nevertheless, crystalluria was present after a 6- hour storage period. CONCLUSION: The tests revealed that cooling preserved cellularity and chemical characteristics of urine samples for up to 12 hours. Nonetheless, the precipitation of crystals was evident in this storage method. Thus, the possible consequences of storing urine samples for ACS test under these conditions should be included in the analysis report.

Effect of Long-term Storage of Urine Samples on Measurement of Kidney Injury Molecule 1 (KIM-1) and Neutrophil Gelatinase-Associated Lipocalin (NGAL)

Levels of urinary biomarkers, such as kidney injury molecule 1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL), can determine the risk of renal damage and predict the outcome of multiple kidney diseases, including transplant rejection. Studies of urinary biomarkers frequently use urine that has been stored frozen before analysis without taking into account the potential of biomarker degradation during storage. Observational study. 39 kidney transplant patients visiting an outpatient clinic. Urine storage conditions (4°C and-80°C with or without protease inhibitors) and centrifugation after thawing of samples. Urinary concentrations of NGAL and KIM-1. NGAL and KIM-1 were measured with commercially available enzyme-linked immunosorbent assay kits. Urinary NGAL and KIM-1 concentrations were stable in urine up to 48 hours when stored at 4°C and up to 6 months when stored at-80°C, independent of the addition of protease inhibitors. A centrifugation step prior to biomarker measurement did not change urinary biomarker concentrations of urine stored at-80°C. Only KIM-1 and NGAL were measured, which possibly limits the relevance of the findings when making determinations about other urinary biomarkers. This study recommends that urine samples collected for the assessment of KIM-1 or NGAL are immediately cooled to 4°C and subsequently frozen at-80°C within 2 days. At-80°C, both KIM-1 and NGAL concentrations are stable for at least 6 months.

Long-Term Frozen Storage of Urine Samples: A Trouble to Get PCR Results in Schistosoma spp. DNA Detection?

BackgroundHuman schistosomiasis remains a serious worldwide public health problem. At present, a sensitive and specific assay for routine diagnosis of schistosome infection is not yet available. The potential for detecting schistosome-derived DNA by PCR-based methods in human clinical samples is currently being investigated as a diagnostic tool with potential application in routine schistosomiasis diagnosis. Collection of diagnostic samples such as stool or blood is usually difficult in some populations. However, urine is a biological sample that can be collected in a non-invasive method, easy to get from people of all ages and easy in management, but as a sample for PCR diagnosis is still not widely used. This could be due to the high variability in the reported efficiency of detection as a result of the high variation in urine samples’ storage or conditions for handling and DNA preservation and extraction methods.Methodology/Principal FindingsWe evaluate different commercial DNA extraction methods from a series of long-term frozen storage human urine samples from patients with parasitological confirmed schistosomiasis in order to assess the PCR effectiveness for Schistosoma spp. detection. Patientś urine samples were frozen for 18 months up to 7 years until use. Results were compared with those obtained in PCR assays using fresh healthy human urine artificially contaminated with Schistosoma mansoni DNA and urine samples from mice experimentally infected with S. mansoni cercariae stored frozen for at least 12 months before use. PCR results in fresh human artificial urine samples using different DNA based extraction methods were much more effective than those obtained when long-term frozen human urine samples were used as the source of DNA template.Conclusions/SignificanceLong-term frozen human urine samples are probably not a good source for DNA extraction for use as a template in PCR detection of Schistosoma spp., regardless of the DNA method of extraction used.

Effects of Storage Temperature, pH and Time on Urinary Albumin Level

The storage of urine samples at 2-8 o C and at -20 o C for several weeks is a common laboratory practice in research and epidemiological studies to facilitate batch analysis. Despite numerous studies, there is no agreement on how stable albumin is in urine and the best way to store such urine specimen. Random urine samples were obtained from 15 patients with nephropathy and end stage renal failure. Urinary pH and specific gravity of freshly voided urine were determined before storage. A portion of each urine samples were stored at voided pH and another portion stored at neutral pH (7.0) after adjustment with acid or base at 4 0 C and -20 0 C for 10 weeks. Pre -analytical treatment of samples involved vortex mixing or centrifuging urine samples before measurement of urinary albumin. Albumin levels were determined at 0, 2, 4 and 10 weeks in both vortex mixed and centrifuged samples using standard spectrophotometric methods. Mean pH and specific gravity of fresh urine samples were 5.82 ± 0.71 and 1.009±0.004 respectively. Significant decreases (p< 0.05) were observed in urinary albumin levels at 4 0 C at the 4 th week. Significant changes were seen at the 10 th week in samples stored at -20 0 C in pH unadjusted samples. Vortex mixing or centrifuging of sample of did not restore decreases in albumin level. No significant difference was observed in the pH adjusted group after 10 weeks of storage at -20 o C.These results suggest that where long term storage (6-10 weeks) of urine samples is required, samples should be stored at -20 0 C. However, in medium term storage (2-4 weeks) storage at 4 0 C may require the adjustment of the pH to neutral (7.0) before storage to obtain reliable results. Keywords: Urinary albumin, temperature, pH, time, storage

Study of volatile selenium metabolites stability in normal urine: effects of sample handling and storage conditions

The aim of this study was to define suitable conditions for sample handling and storage of urine samples in order to ensure reliable analysis of selenium-containing volatile metabolites. Short-term stability testing (from sample collection to 30–35 hours) included: temperature (room, i.e. 22 ± 2 °C, refrigerator (4 °C)), the presence and absence of light, and addition of a bactericide (0.05% NaN3). The long-term stability study evaluated urine storage at −20 and −80 °C over a period of 4 weeks. This work was conducted with normal urine samples, i.e. from non-supplemented human volunteers, in order to profile baseline metabolites and corresponding concentration levels. The results indicate that urine samples should be maintained in the dark immediately after collection and preferably analyzed within the day of collection. If temporary storage cannot be avoided, it should be at 4 °C after addition of NaN3. For longer period, urine samples can be stored at −20 or −80 °C without measurable changes respectively for 2 to 4 weeks.

Development and Validation of a Direct LC-MS-MS Method to Determine the Acrolein Metabolite 3-HPMA in Urine

A new direct method using liquid chromatography-tandem mass spectrometry has been developed and validated for quantitation of 3-hydroxypropylmercapturic acid (3-HPMA) in urine. The method is fast, simple, and does not require extraction from urine. Analyte was separated on a hydrophilic interaction liquid chromatography column. Severe ion suppression was circumvented by a fast gradient after separation. Assay specificity, linearity, precision, and accuracy met the required FDA/CDER bioanalytical method criteria. Matrix effect and carryover of the assay were assessed. Urine sample storage stability and standard solution stability were also tested. The limit of quantitation was 22.0 ng/mL. The results for 3-HPMA obtained by our method were significantly correlated with results obtained by a contract lab.

Quantitation of isoprostane isomers in human urine from smokers and nonsmokers by LC-MS/MS

A simple, rapid liquid chromatography-tandem mass spectrometry method was developed to identify and quantitate in human urine the isoprostanes iPF(2 alpha)-III, 15-epi-iPF(2 alpha)-III, iPF(2 alpha)-VI, and 8,12-iso-iPF(2 alpha)-VI along with the prostaglandin PGF(2 alpha) and 2,3-dinor-iPF(2 alpha)-III, a metabolite of iPF(2 alpha)-III. Assay specificity, linearity, precision, and accuracy met the required criteria for most analytes. The urine sample storage stability and standard solution stability were also tested. The methodology was applied to analyze 24 h urine samples collected from smokers and nonsmokers on controlled diets. The results for iPF(2 alpha)-III obtained by our method were significantly correlated with results by an ELISA, although an approximately 2-fold high bias was observed for the ELISA data. For iPF(2 alpha)-III and its metabolite 2,3-dinor-iPF(2 alpha)-III, smokers had significantly higher concentrations than nonsmokers (513 +/- 275 vs. 294 +/- 104 pg/mg creatinine; 3,030 +/- 1,546 vs. 2,046 +/- 836 pg/mg creatinine, respectively). The concentration of iPF(2 alpha)-VI tended to be higher in smokers than in nonsmokers; however, the increase was not statistically significant in this sample set. Concentrations of the other three isoprostane isomers showed no trends toward differences between smokers and nonsmokers. Among smokers, the daily output of two type VI isoprostanes showed a weak correlation with the amount of tobacco smoke exposure, as determined by urinary excretion of total nicotine equivalents.

Long-term stability of various drugs and metabolites in urine, and preventive measures against their decomposition with special attention to filtration sterilization

The long-term stability of drugs and metabolites of forensic interest in urine, and preventive measures against their decomposition have been investigated, with special attention to filtration sterilization. An aseptic urine collection kit, which was recently developed based on filtration sterilization, was utilized for the aseptic collection and storage of urine samples. For evaluating preservation measures, methamphetamine (MA), amphetamine (AP), nitrazepam (NZ), estazolam (EZ), 7-aminoflunitrazepam (7AF), cocaine (COC), and 6-acetylmorphine (6AM) were spiked into urine at 500 ng/mL each, and were monitored for 6 months at 25, 4, and −20 °C, after the addition of NaN 3 and/or filtration sterilization using the aseptic collection kit. In severely contaminated urine with bacteria, there were significant losses of 7AF and NZ, and slight decomposition of MA and AP at 25 °C. However, such degradation was successfully suppressed by the use of the kit, though the use of the kit and NaN 3 were preferred for 7AF. The kit was also effective in preventing the hydrolyses of COC and 6AM, while it was suggested that the common preservative NaN 3 can accelerate the hydrolysis of such ester-type drugs and metabolites.

Prolonged Frozen Storage of Urine Reduces the Value of Albuminuria for Mortality Prediction

Albuminuria is increasingly recognized as a cardiovascular risk factor in patients with diabetes and in the general population. Cardiovascular disease risk increases continuously with increasing urinary albumin excretion, starting at concentrations that once were considered healthy (1). We recently reported that, after prolonged frozen storage of urine samples, albumin concentrations may decrease, particularly those within the reference and microalbuminuric intervals(2). We also observed sample variation in the extent to which urinary albumin concentrations (UAC) decreased. In 2005, to investigate whether outcome predictions based on albuminuria were affected by assessment of UAC from frozen samples, we reassessed UAC in Prevention of Renal and Vascular ENdstage Disease (PREVEND) study baseline urine samples, collected and stored frozen at −20 °C from 1997 to 1998. The PREVEND study was approved by the local medical ethics committee and conducted in accordance with the guidelines of the Declaration of Helsinki(3). The PREVEND dataset was enriched for albuminuria. To create a representative sample of the general population, the enriched subset (UAC >10 mg/L) was …

Analysis of pyrimidine synthesis "de novo" intermediates in urine and dried urine filter- paper strips with HPLC-electrospray tandem mass spectrometry.

The concentrations of the pyrimidine "de novo" metabolites and their degradation products in urine are useful indicators for the diagnosis of an inborn error of the pyrimidine de novo pathway or a urea-cycle defect. Until now, no procedure was available that allowed the analysis of all of these metabolites in a single analytical run. We describe a rapid, specific method to measure these metabolites by HPLC-tandem mass spectrometry. Urine or urine-soaked filter-paper strips were used to measure N-carbamyl-aspartate, dihydroorotate, orotate, orotidine, uridine, and uracil. Reversed-phase HPLC was combined with electrospray ionization tandem mass spectrometry, and detection was performed by multiple-reaction monitoring. Stable-isotope-labeled reference compounds were used as internal standards. All pyrimidine de novo metabolites and their degradation products were measured within a single analytical run of 14 min with lower limits of detection of 0.4-3 micromol/L. The intra- and interassay variation for urine with added compounds was 1.2-5% for urines and 2-9% for filter-paper extracts of the urines. Recoveries of the added metabolites were 97-106% for urine samples and 97-115% for filter-paper extracts of the urines. Analysis of urine samples from patients with a urea-cycle defect or pyrimidine degradation defect showed an aberrant metabolic profile when compared with controls. HPLC with electrospray ionization tandem mass spectrometry allows rapid testing for disorders affecting the pyrimidine de novo pathway. The use of filter-paper strips could facilitate collection, transport, and storage of urine samples.

Direct quantification of ethyl glucuronide in clinical urine samples by liquid chromatography-mass spectrometry.

Ethyl glucuronide is a minor metabolite of ethanol, and its presence in urine can be used as a laboratory test to detect recent alcohol intake, even for some time after the ethanol is no longer measurable. A simple analytical procedure was developed based on direct injection of urine diluted with a deuterated internal standard into an electrospray liquid chromatographic-mass spectrometric (LC-MS) system. A novel LC system using a porous graphite column (Hypercarb) enabled an isocratic elution with retention times of 5-6 minutes. The intra- and inter-assay coefficients of variation were 2-12%, and the measuring range was 0.1-1,500 mg/L (0.45-6,750 micromol/L). Ethyl glucuronide was found to be stable in urine for more than 4 days at room temperature, and no artifactual formation was observed on storage of urine samples fortified with 1% ethanol. Ethyl glucuronide was not detected in urine samples collected after abstinence from alcohol. Intake of a very low amount (7 g) of ethanol produced ethyl glucuronide values up to 8.4 mg/L after 4 hours and was still detectable at 6 hours. When the method was applied for routine screening of 252 clinical urine samples (range, 0-1,240 mg/L), it fulfilled the need for a simple and reliable assay to be used in the evaluation of urinary ethyl glucuronide as a routine test of recent alcohol intake.

Short-Term Stability of Lysergic Acid Diethylamide (LSD), N-Desmethyl-LSD, and 2-Oxo-3-hydroxy-LSD in Urine, Assessed by Liquid Chromatography–Tandem Mass Spectrometry

Lysergic acid diethylamide (LSD) is one of the most potent hallucinogenic agents known. Recently, data on emergency department episodes related to the use of drugs commonly thought as “club drugs” have also included LSD (1). Confirmation of LSD use by testing biological fluids is still an analytical challenge because of its extensive, rapid metabolism and its instability (2)(3)(4). After ingestion of a typical street dose (40–120 μg), the concentration of LSD in urine falls to <1 μg/L within a few hours (2)(5)(6). Recently, N -desmethyl-LSD (nor-LSD) and 2-oxo-3-hydroxy-LSD (O-H-LSD) have been identified as LSD metabolites in human urine (7)(8). Measured nor-LSD concentrations were reported to be in the same concentration range as LSD, whereas the measured concentrations of O-H-LSD were severalfold higher (0.02–21.4 μg/L) (7)(9)(10)(11). The application of liquid chromatography–tandem mass spectrometry (LC-MS/MS) improved the detection of LSD use (9)(10)(12)(13)(14)(15); the method is also less susceptible to interferences when used for drug screening (16)(17). In a few studies, O-H-LSD and nor-LSD have also been measured (6)(7)(8)(10)(12)(13)(14)(15). LSD has been shown to rapidly decompose in urine samples exposed to an increased temperature or to sunlight or ultraviolet light (18)(19)(20)(21), but stability data on major LSD metabolites are not yet available. This study was undertaken to determine the stability of LSD, O-H-LSD, and nor-LSD in urine under different storage conditions by LC-MS/MS. Data on the reaction order type and major influencing factors may be useful in planning transport and storage of urine samples. Stability was assessed by enriching a drug-free urine sample from a …

Study of the stability of selenium compounds in human urine and determination by mixed ion-pair reversed-phase chromatography with ICP-MS detection.

The stability of five selenium compounds, selenate, Se(VI), selenourea, SeUr, trimethylselenonium ion, TMSe(+), selenomethionine, SeMet, and selenoethionine, SeEt, at concentrations from 30-60 micro g L(-1) in a pooled human urine, stored in dark at -20 degrees C, 4 degrees C, or ambient temperature (ca. 25 degrees C), without addition of any stabilizing reagent was evaluated. The investigated Se species were determined independently by mixed ion-pair reversed-phase liquid chromatography with inductively coupled plasma mass spectrometric (ICP-MS) detection. The general trend is the lower the temperature used for storage, the higher the stability of Se species, when other conditions such as light, acidity, and container material are kept constant. On the basis of these results it is considered that the storage of urine samples at -20 degrees C for a short-term (within one month) is safe for Se speciation analysis. Long-term storage of urine samples for speciation analysis should, however, be undertaken with caution.

Defects in Pyrimidine Degradation Identified by HPLC-Electrospray Tandem Mass Spectrometry of Urine Specimens or Urine-soaked Filter Paper Strips

Urinary concentrations of thymine, uracil, and their degradation products are useful indicators of deficiencies of enzymes of the pyrimidine degradation pathway. We describe a rapid, specific method to measure these concentrations to detect inborn errors of pyrimidine metabolism. We used urine or urine-soaked filter-paper strips as samples and measured thymine, uracil, and their degradation products dihydrothymine, dihydrouracil, N:-carbamyl-ss-aminoisobutyric acid, and N:-carbamyl-ss-alanine. Reversed-phase HPLC was combined with electrospray ionization tandem mass spectrometry, and detection was performed by multiple-reaction monitoring. Stable-isotope-labeled reference compounds were used as internal standards. All pyrimidine degradation products could be measured in one analytical run of 15 min. Detection limits were 0.4-4 micromol/L. The intraassay imprecision (CV) of urine samples with added compounds was 1.3-12% for liquid urines and 1. 0-10% for filter-paper extracts of the urines. The interassay imprecision (CV) was 3-11% (100-200 micromol/L). Recoveries were 89-99% at 100-200 micromol/L and 95-106% at 1 mmol/L in liquid urines, and 93-103% at 100-200 micromol/L and 100-106% at 1 mmol/L in filter-paper samples. Correct identifications of deficiencies of the pyrimidine-degrading enzymes were readily made with urine samples from patients with known defects. HPLC with electrospray ionization tandem mass spectrometry allows rapid testing for disorders of the pyrimidine degradation pathway, and filter-paper samples allow easy collection, transport, and storage of urine samples.

Sample Preparation and Storage Can Change Arsenic Speciation in Human Urine

Stability of chemical speciation during sample handling and storage is a prerequisite to obtaining reliable results of trace element speciation analysis. There is no comprehensive information on the stability of common arsenic species, such as inorganic arsenite [As(III)], arsenate [As(V)], monomethylarsonic acid, dimethylarsinic acid, and arsenobetaine, in human urine. We compared the effects of the following storage conditions on the stability of these arsenic species: temperature (25, 4, and -20 degrees C), storage time (1, 2, 4, and 8 months), and the use of additives (HCl, sodium azide, benzoic acid, benzyltrimethylammonium chloride, and cetylpyridinium chloride). HPLC with both inductively coupled plasma mass spectrometry and hydride generation atomic fluorescence detection techniques were used for the speciation of arsenic. We found that all five of the arsenic species were stable for up to 2 months when urine samples were stored at 4 and -20 degrees C without any additives. For longer period of storage (4 and 8 months), the stability of arsenic species was dependent on urine matrices. Whereas the arsenic speciation in some urine samples was stable for the entire 8 months at both 4 and -20 degrees C, other urine samples stored under identical conditions showed substantial changes in the concentration of As(III), As(V), monomethylarsonic acid, and dimethylarsinic acid. The use of additives did not improve the stability of arsenic speciation in urine. The addition of 0.1 mol/L HCl (final concentration) to urine samples produced relative changes in inorganic As(III) and As(V) concentrations. Low temperature (4 and -20 degrees C) conditions are suitable for the storage of urine samples for up to 2 months. Untreated samples maintain their concentration of arsenic species, and additives have no particular benefit. Strong acidification is not appropriate for speciation analysis.

Development and validation of an assay for urinary tissue factor activity.

BACKGROUND: Activation of blood coagulation is a common complication of cancer and inflammation in both humans and experimental animals. Increased production of tissue factor--the principal initiator of the coagulation process--by...

A 31P NMR Study of the Stability of Carboxyifosfamide and Carboxycyclophosphamide, Two Metabolites of Ifosfamide and Cyclophosphamide

Abstract Ifosfamide (IF) and cyclophosphamide (CP) are two phosphorated anticancer agents used in the treatment of solid tumours. Several phosphorated metabolites, among them carboxyifosfamide (CXIF) and carboxycyclophosphamide (CXCP), were detected and quantified by 31P NMR in urine from patients treated with IF or CP. In agreement with other authors [1], we observed a great inter-patient variability in the urinary excretion of CXIF in patients treated with IF [2]. This variability was attributed to a genetic polymorphism of aldehyde dehydrogenase, the enzyme responsible for the formation of CXCP or CXIF [1,3]. Since CXCP and CXIF are unstable, we thought that the inter-individual variability could also be due to a degradation during the storage of urine samples. A 31P NMR study of the stability of CXIF and CXCP in urine as a function of time, pH (7 and 5.5) and storage temperature (25°C, 8°C, −20°C, −80°C) demonstrated that (i) CXCP and CXIF are more stable at pH 7 than at pH 5.5, (ii) CXCP is more stab...

Frozen storage of urine samples before ELISA measurement of retinol-binding protein

Frozen storage of urine samples before ELISA measurement of retinol-binding protein