Osmolality-based normalization enhances statistical discrimination of untargeted metabolomic urine analysis: results from a comparative study.

INTRODUCTION: Because of its ease of collection, urine is one of the most commonly used matrices for metabolomics studies. However, unlike other biofluids, urine exhibits tremendous variability that can introduce confounding inconsistency during result interpretation. Despite many existing techniques to normalize urine samples, there is still no consensus on either which method is most appropriate or how to evaluate these methods. OBJECTIVES: To investigate the impact of several methods and combinations of methods conventionally used in urine metabolomics on the statistical discrimination of two groups in a simple metabolomics study. METHODS: We applied 14 different strategies of normalization to forty urine samples analysed by liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS). To evaluate the impact of these different strategies, we relied on the ability of each method to reduce confounding variability while retaining variability of interest, as well as the predictability of statistical models. RESULTS: Among all tested normalization methods, osmolality-based normalization gave the best results. Moreover, we demonstrated that normalization using a specific dilution prior to the analysis outperformed post-acquisition normalization. We also demonstrated that the combination of various normalization methods does not necessarily improve statistical discrimination. CONCLUSIONS: This study re-emphasized the importance of normalizing urine samples for metabolomics studies. In addition, it appeared that the choice of method had a significant impact on result quality. Consequently, we suggest osmolality-based normalization as the best method for normalizing urine samples. TRIAL REGISTRATION NUMBER: NCT03335644.

Accuracy of urine flow cytometry and urine test strip in predicting relevant bacteriuria in different patient populations.

BACKGROUND: Urinary tract infection (UTI) is diagnosed combining urinary symptoms with demonstration of urine culture growth above a given threshold. Our aim was to compare the diagnostic accuracy of Urine Flow Cytometry (UFC) with urine test strip in predicting bacterial growth and in identifying contaminated urine samples, and to derive an algorithm to identify relevant bacterial growth for clinical use. METHODS: Species identification and colony-forming unit (CFU/ml) quantification from bacterial cultures were matched to corresponding cellular (leucocytes/epithelial cells) and bacteria counts per µl. Results comprise samples analysed between 2013 and 2015 for which urine culture (reference standard) and UFC and urine test strip data (index tests, Sysmex UX-2000) were available. RESULTS: 47,572 urine samples of 26,256 patients were analysed. Bacteria counts used to predict bacterial growth of ≥105 CFU/ml showed an accuracy with an area under the receiver operating characteristic curve of > 93% compared to 82% using leukocyte counts. The relevant bacteriuria rule-out cut-off of 50 bacteria/µl reached a negative predictive value of 98, 91 and 89% and the rule-in cut-off of 250 bacteria/µl identified relevant bacteriuria with an overall positive predictive value of 67, 72 and 73% for microbiologically defined bacteriuria thresholds of 105, 104 or 103 CFU/ml, respectively. Measured epithelial cell counts by UFC could not identify contaminated urine. CONCLUSIONS: Prediction of a relevant bacterial growth by bacteria counts was most accurate and was a better predictor than leucocyte counts independently of the source of the urine and the medical specialty ordering the test (medical, surgical or others).

Are individual analyses of multiple short urine collections throughout the 24 hours superior to a standard 24-hour urine collection in precipitation risk assessment of healthy subjects?

PURPOSE: The commonly used 24-hour collection technique has been the mainstay of diagnosis for supersaturation but has some certain limitations. Hence, superiority of multiple short urine collections as a new alternative in precipitation risk assessment was assessed compared to the standard 24-hour urine collection among healthy subjects. MATERIALS AND METHODS: Individual urine samples of 26 healthy subjects were acquired every 2 to 3 hours throughout the 24 hours. Urine samples were obtained and the time and volume of each sample were recorded. Urinary constituents involved in precipitation including, sodium-potassium, chloride, calcium, phosphate, citrate, magnesium, urea, creatinine and pH were measured. A simulated 24-hour collection was recalculated by the totalling of all shorter urine collections volume and urinary constituents excretions throughout the day. RESULTS: Urine pH, urine creatinine and precipitation rate had a significantly lower values in 24-hours urine collection compared to one individual value of multiple urine collections by -0.769 (P < .0001), -7.305 (P < .0001), and - 12.838 (P < .0001), respectively. However, calcium (2.697, P < .0001), citrate (3.54, P < .0001), total phosphate (19.961, P < .0001) and total creatinine (9.579, P < .0001) had statistically significantly higher values in the 24-hours urine collection compared to individual value of multiple urine collections. CONCLUSION: Based on the results, individual analysis of multiple shorter urine collections throughout the day improves the ability of identifying supersaturation points, precipitation risk zones and may potentially improve risk assessment compared to the 24-hour urine collection method.

Reference values of urine protein/creatinine ratio in healthy Dalian adults.

BACKGROUND: The urine protein/creatinine ratio (UPCR) is commonly used in current clinical practice. However, there are only few published clinical data on UPCR from large cohorts of Chinese adults. This study aimed to determine the overall and age- and sex-specific UPCR reference values for healthy Dalian adults. METHODS: According to the Clinical & Laboratory Standards Institute EP28-A3c guidelines, 1321 healthy Dalian adults (646 men and 675 women) aged 20-69 years were enrolled. Urine protein and creatinine levels were analyzed in the random morning spot urine samples, and UPCR was calculated. The 95th percentile of the UPCR was used as the normal upper limit. The Mann-Whitney U test was used to test differences among groups. RESULTS: The UPCR reference value was 141.7 mg/g for the entire cohort, 128.7 mg/g for men, and 150.8 mg/g for women. In addition, women had relatively higher UPCR values than men in the same age group. We also compared the UPCR reference values between different estimated glomerular filtration rate (eGFR) groups and found that women had significantly higher UPCR values than men in the normal eGFR groups. CONCLUSIONS: This study provides the overall and age- and sex-specific UPCR reference values for healthy Dalian adults.

Application of a six sigma model to evaluate the analytical performance of urinary biochemical analytes and design a risk-based statistical quality control strategy for these assays: A multicenter study.

BACKGROUND: The six sigma model has been widely used in clinical laboratory quality management. In this study, we first applied the six sigma model to (a) evaluate the analytical performance of urinary biochemical analytes across five laboratories, (b) design risk-based statistical quality control (SQC) strategies, and (c) formulate improvement measures for each of the analytes when needed. METHODS: Internal quality control (IQC) and external quality assessment (EQA) data for urinary biochemical analytes were collected from five laboratories, and the sigma value of each analyte was calculated based on coefficients of variation, bias, and total allowable error (TEa). Normalized sigma method decision charts for these urinary biochemical analytes were then generated. Risk-based SQC strategies and improvement measures were formulated for each laboratory according to the flowchart of Westgard sigma rules, including run sizes and the quality goal index (QGI). RESULTS: Sigma values of urinary biochemical analytes were significantly different at different quality control levels. Although identical detection platforms with matching reagents were used, differences in these analytes were also observed between laboratories. Risk-based SQC strategies for urinary biochemical analytes were formulated based on the flowchart of Westgard sigma rules, including run size and analytical performance. Appropriate improvement measures were implemented for urinary biochemical analytes with analytical performance lower than six sigma according to the QGI calculation. CONCLUSIONS: In multilocation laboratory systems, a six sigma model is an excellent quality management tool and can quantitatively evaluate analytical performance and guide risk-based SQC strategy development and improvement measure implementation.

Evaluation of conductivity-based osmolality measurement in urine using the Sysmex UF5000.

BACKGROUND: Automated flow cytometry-based urine analyzer is increasingly being used to identify and enumerate cells and particles in urine specimens. It measures electrical conductivity which could be transformed to osmolality. Using this machine, all urine specimens could be screened for osmolality without requiring a separate dedicated device. We evaluated the performance of the new instrument, the UF-5000 (Sysmex Corporation), in the measurement of urine osmolality. METHODS: The precision of urine osmolality measurement by the UF-5000 was evaluated for 20 days and 4 times a day for 2 concentrations. The linearity and detection capability were evaluated according to the Clinical and Laboratory Standards Institute guidelines. For comparison, 270 random urine specimens from patients were tested simultaneously using the UF5000 and the OsmoPro micro-osmometer (Advanced instruments). RESULTS: The laboratory-based coefficient variations were less than 5%. Urine osmolality using the UF-5000 has a verified linear range (y = 1.097x + 16.91, R2  = .997). Within the comparison analysis, the mean difference was not large (-7.72%) but each differences were largely dispersed with 95% limits of agreement (LoA) from -70.5 to 55.06%, and the mean absolute difference -28.3 mOsm/kg with 95% LoA from -295.13 to 238.45 mOsm/kg. Cohen's kappa value was 0.54 (95% CI, 0.45-0.63). CONCLUSIONS: The UF-5000 measured conductivity and generated an acceptable quantitative analysis of urine osmolality. When compared with the results of the freezing point depression method used by the OsmoPro, a percentage of the measured urine osmolality by the UF-5000 was outside the allowable limit.

Application of Bar Adsorptive Microextraction for the Determination of Levels of Tricyclic Antidepressants in Urine Samples.

This work entailed the development, optimization, validation, and application of a novel analytical approach, using the bar adsorptive microextraction technique (BAµE), for the determination of the six most common tricyclic antidepressants (TCAs; amitriptyline, mianserin, trimipramine, imipramine, mirtazapine and dosulepin) in urine matrices. To achieve this goal, we employed, for the first time, new generation microextraction devices coated with convenient sorbent phases, polymers and novel activated carbons prepared from biomaterial waste, in combination with large-volume-injection gas chromatography-mass spectrometry operating in selected-ion monitoring mode (LVI-GC-MS(SIM)). Preliminary assays on sorbent coatings, showed that the polymeric phases present a much more effective performance, as the tested biosorbents exhibited low efficiency for application in microextraction techniques. By using BAµE coated with C18 polymer, under optimized experimental conditions, the detection limits achieved for the six TCAs ranged from 0.2 to 1.6 µg L-1 and, weighted linear regressions resulted in remarkable linearity (r2 > 0.9960) between 10.0 and 1000.0 µg L-1. The developed analytical methodology (BAµE(C18)/LVI-GC-MS(SIM)) provided suitable matrix effects (90.2-112.9%, RSD ≤ 13.9%), high recovery yields (92.3-111.5%, RSD ≤ 12.3%) and a remarkable overall process efficiency (ranging from 84.9% to 124.3%, RSD ≤ 13.9%). The developed and validated methodology was successfully applied for screening the six TCAs in real urine matrices. The proposed analytical methodology proved to be an eco-user-friendly approach to monitor trace levels of TCAs in complex urine matrices and an outstanding analytical alternative in comparison with other microextraction-based techniques.

A new point-of-care test for the rapid detection of urinary tract infections.

Urinary tract infections (UTIs) are among the most common infections in all age groups. Fast and accurate diagnosis is essential to ensure a timely and effective therapy. Alongside with reference culture-based methods, several point-of-care tests (POCTs) for early detection of UTIs have been developed, but they have not been significantly implemented in current clinical practice. The Micro Biological Survey (MBS) POCT is a simple test developed by MBS Diagnostics Ltd. (London, UK) for the detection and management of UTIs. The present study has been undertaken to investigate the potentials and limits of the MBS POCT. A total of 349 patients were enrolled in two open-label, monocentric, non-interventional clinical trials in collaboration with an Emergency Medicine department and the outpatient clinic of two hospitals in Rome. Results of urine analysis using the MBS POCT were compared with those of the routine culture-based tests for UTI diagnosis performed by the hospital laboratory. The MBS POCT provided fast results revealing high bacterial count UTIs (≥ 105 CFU/ml) with 97% accuracy, 92% sensitivity, 100% specificity, 99% PPV, and 96% NPV within a 5-h analytical time threshold.

Sample size and rejection limits for detecting reagent lot variability: analysis of the applicability of the Clinical and Laboratory Standards Institute (CLSI) EP26-A protocol to real-world clinical chemistry data.

Objectives: To maintain the consistency of laboratory test results, between-reagent lot variation should be verified before using new reagent lots in clinical laboratory. Although the Clinical and Laboratory Standards Institute (CLSI) document EP26-A deals with this issue, evaluation of reagent lot-to-lot difference is challenging in reality. We aim to investigate a practical way for determining between-reagent lot variation using real-world data in clinical chemistry. Methods: The CLSI EP26-A protocol was applied to 83 chemistry tests in three clinical labs. Three criteria were used to define the critical difference (CD) of each test as follows: reference change value and total allowable error, which are based on biological variation, and acceptable limits by external quality assurance agencies. The sample size and rejection limits that could detect CD between-reagent lots were determined. Results: For more than half of chemistry tests, reagent lot-to-lot differences could be evaluated using only one patient sample per decision level. In many cases, the rejection limit that could detect reagent lot-to-lot difference with ≥90% probability was 0.6 times CD. However, the sample size and rejection limits vary depending on how the CD is defined. In some cases, impractical sample size or rejection limits were obtained. In some cases, information on sample size and rejection limit that met intended statistical power was not found in EP26-A. Conclusions: The CLSI EP26-A did not provide all necessary answers. Alternative practical approaches are suggested when CLSI EP26-A does not provide guidance.

Can methods based on spot urine samples be used to estimate average population 24 h sodium excretion? Results from the Isfahan Salt Study.

OBJECTIVE: To assess agreement between established methods of estimating salt intake from spot urine collections and 24 h urinary Na (24hUNa) and then to develop a valid formula that can be used in the Iranian population to estimate salt intake from spot urine samples. DESIGN: A validation study. Three spot urine samples were collected (fasting second-void morning; afternoon; evening) on the same day as a 24 h urine collection. We estimated 24hUNa from spot specimens using the Kawasaki, Tanaka and INTERSALT equations. Two new formulas were developed, the Iran formula 1 (Iran 1) and Iran formula 2 (Iran 2), based on our population characteristics. SETTING: Iranian adults recruited in 2014-2015. PARTICIPANTS: Healthy volunteer adults aged ≥18 years. RESULTS: With all three spot urine specimens, predicted population 24hUNa was underestimated based on the INTERSALT equation (-469 to -708 mg/d; all P < 0·05) and conversely overestimation occurred with the Kawasaki equation (926 to 1080 mg/d; all P < 0·01). The Tanaka equation produced comparable estimates to measured 24hUNa (-151 to 86 mg/d; all P > 0·49). The newly derived formulas, Iran 1 and Iran 2, showed less mean bias than the established equations (Iran 1: 43 to 80 mg/d, all P > 0·55; Iran 2: 22 to 90 mg/d, all P > 0·50). CONCLUSIONS: In this Iranian sample, the Tanaka equation and newly derived formulas produced group-level estimates comparable to measured 24hUNa. The newly developed formulas showed less mean bias than established equations; however, they need to be tested for generalization in a larger sample.

A prospective study to assess the association of body mass index and contamination of urinalysis samples.

BACKGROUND: There is a commonly held belief that overweight women are more likely to offer contaminated urine samples (UAs) in the emergency department (ED) than women with normal body mass index (BMI). However, there is a paucity of research evaluating this potential concern. OBJECTIVE: We hypothesized that patients with higher BMI would be more likely to provide contaminated urine samples than women with low BMI. METHODS: This was a prospective, observational, cohort study evaluating consenting, adult, women that provided a clean catch, mid-stream sample at an inner-city ED. UAs were ordered at the discretion of the caring physician, cultures based on standardized parameters. The primary outcome parameter was the presence of UA contamination as defined by our microbiology lab. Demographic/historical data and BMI were recorded on a structured data sheet. Categorical data were analyzed by chi-square; continuous data by t-tests. Multivariable logistic regression was performed to control for confounding. RESULTS: There were 350 patients in the study group; 22% overweight, 35% obese, 17% morbidly obese, mean BMI 31. 5, and 60% provided contaminated specimens. The mean BMIs of the subjects with contaminated vs. uncontaminated UAs were significantly different (32.7 ±â€¯10.2 vs 29.7 ±â€¯8.8, p < 0.01). Within our multiple variable logistic regression model, obese and morbidly obese patients were more likely to provide contaminated UAs, while there were no significant associations for contamination with other variables except for hypertension (OR = 1.85, p = 0.02). CONCLUSION: Obesity was significantly associated with contamination of clean catch mid-stream samples in our population.

Evaluation of WASPLab Software To Automatically Read chromID CPS Elite Agar for Reporting of Urine Cultures.

Urine cultures are among the most common specimens received by clinical laboratories and generate a major share of the laboratory workload. Chromogenic agar can expedite culture results, but technologist review is still needed. In this study, we evaluated the ability of the WASPLab software to interpret urine specimens plated onto chromID CPS Elite (CPSE) agar. Urine specimens submitted for bacterial culture were plated onto CPSE agar with a 1-µl loop using the WASP. Each plate was imaged after 0 and 18 h of incubation, and colonies were enumerated by color using the WASPLab software and a technologist's reading from a high-definition (HD) monitor. The results were reported as negative if <10 colonies/plate were detected. Laboratory information system (LIS) time stamps were used to measure the time to result. A total of 1,581 urine cultures were tested. The sensitivity and specificity of the software were 99.8% and 68.5%, respectively, which included 2 manual-positive/automation-negative (MP/AN) results and 170 manual-negative/automation-positive (MN/AP) results. Of the 170 MN/AP specimens, 116 were caused by microcolonies missed by the technologist. The remaining MN/AP results were caused by either count differences near the 10-colony threshold (n = 43) or count differences of >50 CFU (n = 11). The use of both CPSE agar and the WASPLab software improved the time to result for urine culture, reducing the average time to result by 4 h 42 min for negative specimens and 3 h 28 min for positive specimens compared to that with standard-of-care testing. These data demonstrate that the use of CPSE agar and automated plate reading has the potential to improve turnaround time while maintaining high sensitivity and reducing urine culture workload.

Utility of DNA Next-Generation Sequencing and Expanded Quantitative Urine Culture in Diagnosis and Management of Chronic or Persistent Lower Urinary Tract Symptoms.

Many patients suffer from chronic, irritative lower urinary tract symptoms (LUTS). The evaluation and management of these patients have proven difficult with the use of standard diagnostic tools, including urinalysis and urine culture. The growing body of literature on the urinary microbiome has looked at the possible implications of the bladder microbiome and dysbiosis, or perturbations in the microbiome, in conditions associated with chronic LUTS. Disorders such as recurrent urinary tract infections (UTIs) and interstitial cystitis have been studied utilizing 16S rRNA rapid next-generation gene sequencing (NGS) and expanded quantitative urine culture (EQUC). In this article, we first present a brief review of the literature describing the current understanding of the urinary microbiome and the features and applications of NGS and EQUC. Next, we discuss the conditions most commonly associated with chronic, persistent LUTS and present the limitations of current diagnostic practices utilized in this patient population. We then review the limited data available surrounding treatment efficacy and clinical outcomes in patients who have been managed based on results provided by these two recently established diagnostic tools (DNA NGS and/or EQUC). Finally, we propose a variety of clinical scenarios in which the use of these two techniques may affect patients' clinical outcomes.

Performance of yeast-like cell counting (YLCC) using the Sysmex UF-1000i for clinical candiduria screening.

Candiduria is common in clinical practice. However, an effective and convenient assay to screen for candiduria is still needed. This study aimed to evaluate the performance of the Sysmex UF-1000i urine analyzer for yeast-like cell counting (YLCC) to screen for candiduria prior to urine culture. We retrospectively analyzed data from 5233 urine samples from 1813 patients, including 837 males and 976 females. Urine culture and urinalysis-obtained YLCC data were used to estimate the performance of YLCC in diagnosing candiduria. Different cutoff values were used to calculate sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). The YLCC-positive rates differed according to the Candida colony-forming units (CFU) counts in the urine samples. A sharp drop in YLCC-positive rate (from 64.3 to 22.0%) was observed between the urine groups with 104 CFUs and 103 CFUs. A cutoff value of 0 YLCs/µL results in the highest Youden index (0.71) with 77.04% sensitivity and 93.68% specificity. In a group of 34 hospitalized candiduria patients with serial urinalysis data, 25 were YLCC-positive before urine culture. In conclusion, YLCC with the Sysmax UF-1000i could serve as an auxiliary technique to exclude culture-negative specimens prior to urine culture. Positive YLCC results could imply candiduria, especially when persistent YLCC-positive results were observed.

Establishment of the intelligent verification criteria for a routine urinalysis analyzer in a multi-center study.

Background Although laboratory information system (LIS) is widely used nowadays, the results of routine urinalysis still need 100% manual verification. We established intelligent verification criteria to perform the automated verification process and reduce manual labor. Methods A total of 4610 urine specimens were obtained from the patients of three hospitals in Beijing, China. Firstly, 895 specimens were measured to establish the reference intervals of formed-element parameters in UF5000. Secondly, 2803 specimens were analyzed for setting up the intelligent verification criteria (including the microscopic review rules and manual verification rules). Lastly, 912 specimens were used to verify the efficacy and accuracy of the intelligent verification criteria. Phase-contrast microscopes were used for the microscopic review. Results Employing a results level corresponding relationship in specific parameters including hemoglobin (red blood cell [RBC]), leukocyte esterase (white blood cell [WBC]) and protein (cast) between the dry-chemistry analysis and formed-element analysis, as well as instrument flags, we established seven WBC verification rules, eight RBC verification rules and four cast verification rules. Based on the microscopy results, through analyzing the pre-set rules mentioned earlier, we finally determined seven microscopic review rules, nine manual verification rules and three auto-verification rules. The microscopic review rate was 21.98% (616/2803), the false-negative rate was 4.32% (121/2803), the total manual verification rate was 35.71% (1001/2803) and the auto-verification rate was 64.29% (1802/2803). The validation results were consistent. Conclusions The intelligent verification criteria for urinary dry-chemistry and urinary formed-element analysis can improve the efficiency of the results verification process and ensure the reliability of the test results.

Improving the Detection of Urine Sediment with a Modified Urinalysis Review Procedure.

BACKGROUND: Currently, numerous review procedures are applied to perform the urine sediment examination. Clinical technologists and nephrologists use different procedures for the determination of specimen concentration and for particle counting. These techniques may underestimate the formed elements such as pathological casts (CASTs) and renal tubular epithelial (RTE) cells and might interfere with clinical diagnosis. The aim of this study was to evaluate a modified review procedure for urinary analysis and to narrow the gap between nephrologists and technologists by increasing the detection positivity rate for pathological formed elements. METHODS: We implemented a modified urinalysis procedure between October 2016 and January 2017 based on strict manual microscopic criteria and the currently available equipment. We confirmed the agreement between methods using a review procedure and Sysmex UF-1000i urinary flow cytometer (Pairwise Agreement > 0.88 for WBCs, RBCs, CASTs, and SRCs). Then we derived the review procedure that was based on the optimal sensitivity and specificity as follows: RBC > 26.1/µL, WBC > 37.0/µL, CAST > 1.0/µL, SRC > 8.2/µL, XTAL > 1.5/µL, YLC > 10.0/µL, BACT > 287.5/µL. RESULTS: Of the 317 specimens investigated, 17.4% (26/149) and 31.5% (39/124) of the specimens for RTEs and Path. CASTs, respectively, were correctly detected using the proposed review procedure. Sensitivity and specificity for this procedure was 96.9% and 46.2%, respectively. In addition, we verified the ability of the procedure to detect the pathological elements with technologists and nephrologists and the agreement was satisfactory. CONCLUSIONS: This modified review procedure can significantly improve the quality of urinalysis and reduce the risk of underestimating the detection of pathological particles.

Comparison of Hydrostatic Filtration Dialysis with Ultracentrifugation Methods for the Identification and Proteomic Profiling of Urinary Extracellular Vesicles.

BACKGROUND: Urinary extracellular vesicles (UEVs) carry rich markers of their parent cells, so they can serve as possible biomarkers of kidney diseases. METHODS: In this study, we isolated urinary extracellular vesicles from five individuals using a simple, clinically applicable method called hydrostatic filtration dialysis (HFD) and compared it to the gold-standard ultracentrifuga-tion (UC) with transmission electron microscopy (TEM). We also employed a proteomic approach using pooled human urine samples from the same five individuals to profile the protein composition of UEVs to evaluate the effectiveness of these two methods. RESULTS: Notably, using TEM, we found that all isolations contained 0 - 400 nm vesicles with the traditionally reported morphology, although the TEM results showed that the UEVs isolated from HFD compared to those from UC are larger and more extensive. We obtained a total of 2,564 UEV proteins in the two methods. We showed a large overlap (2,185 > 85%) between the proteins identified by both isolation methods. The result also showed that the obtained proteins in extracellular vesicles, which are isolated with these methods, are consistent with the results in currently available databases. However, in the associated gene ontologies, the enriched proteins found by the two methods showed some differences. CONCLUSIONS: The HFD method is clinically feasible and allows large-scale protein profiling of UEV biomarkers. The results of this study also provide valuable UEV protein data from the methodological comparison, which might be valuable to other researchers.

Establishment of a reference procedure to measure urine-formed elements and evaluation of an automated urine analyzer.

A standardized reference method is needed to accurately and precisely measure urine-formed elements (UFEs; red blood cells [RBCs], white blood cells [WBCs], and squamous epithelial cells [sECs]). We compared the results from a standard method with those from an automated analyzer. Trained technicians used standardized bright-field microscopy of fresh non-centrifuged urine samples, and disposable 1 µl chambers. Fifteen experienced technicians from 5 hospitals (3 per hospital) each performed 6 manual counts of 10 different native urine samples using a manual chamber and standard methods. The sEC counts were at least 50/µL, and the coefficient of variation (CV) was less than 14%; the RBC and WBC counts were at least 200/µL and the CVs were less than 7%. The same samples were also analyzed 6 times using automated analyzers. The means, CVs, and biases were determined. The median CVs for the manual measurements were 6.4% (WBCs), 6.6% (RBCs), and 12.7% (sECs). The CVs of the automated analyzer were 4.7% (WBCs), 5.6% (RBCs), and 9.2% (sECs). Biases between the automated and manual methods were -2.9% to 5.0%(WBCs), -0.8% to 8.8% (RBCs) and -2.8% to 9.4% (sECs). The count mean values and expanded uncertainties of these counts were (224.5 ± 15.0) cells/µL, (234.2 ± 16.2) cells/µL, and (61.5 ± 7.9) cells/µL, respectively. The standardized manual method for measuring UFEs had high precision and accuracy, making it a suitable reference method. Use of this reference method to calibrate an automated analyzer improved the accuracy of automated analysis.

The standardization of the report for urine cell counting-A converting factor for Sysmex UF-1000i.

BACKGROUND: Multicenter laboratory may apply both automated flow cytometer and microscopy for urinalysis. Automated flow cytometer such as Sysmex UF-1000i evaluates particles with native urine without centrifugation and reports as "counts per µL." Microscopic examination recommended as the reference method for urine sediment analysis reports results as "counts per HPF (or µL)." Moreover, some results from flow cytometer are needed to be checked visually under microscopy. Therefore, it is worth to establish the consistency of the results from these two methods. METHODS: Urine specimens from 412 patients were examined with Sysmex UF-1000i and manual microscopy using FAST-READ disposable counting chambers. White blood cell (WBC) and red blood cell (RBC) counting results from UF-1000i after transferred with the converting factor (0.297) we estimated were compared with that from microscopic examination. Method comparison was performed using Passing-Bablok analysis. RESULTS: After transferred with the converting factor (0.297), cell counting results from UF-1000i showed a good correlation with that derived by the reference method (R2 was 0.868 for RBCs (P < 0.001), 0.882 for WBCs (P < 0.001)). Passing-Bablok analysis showed no systematic difference (intercept estimate, -1 [95%CI, -7 to 3] and slightly proportional (slope estimate, 1.2 [95%CI, 1.0 to 1.7]) bias between concentrations of cells measured by manual microscopy and Sysmex UF-1000i using the converting factor. CONCLUSION: The converting factor (0.297) helps to transfer "counts per µL (non-centrifugal urine)" to "counts per µL (equal to centrifugal urine)," and to keep the urine particle analysis results of Sysmex UF-1000i consistent with the results from the reference method.