Comparison of the clinical performance of the Atyp.C parameter of the UF-5000 fully automated urine particle analyzer with that of microscopic urine sediment analysis.

a Objectives: Urinalysis is one of the most common laboratory screening tests to detect problems in the renal and urinary system; however, they cannot detect atypical cells (Atyp.Cs). The Sysmex UF-5000, a fully automated urine particle analyzer, can detect Atyp.Cs via its Atyp.C parameter. This study aimed to compare the clinical value of the Atyp.C parameter with that of urine sediment microscopy. b Method: A total of 471 leftover urine samples were submitted to the Department of Clinical Laboratory at the University of Tokyo Hospital for urinalysis by manual sediment microscopy examination and UF-5000 Atyp.C analysis. c Result: Of 471 submitted samples, 117 were positive for Atyp.Cs by urine sediment and 354 samples were negative. The histological subtypes of the Atyp.Cs included 105 cases of suspected urothelial carcinoma cells, 10 suspected squamous carcinoma cells, and 2 of suspected adenocarcinoma cells. The Atyp.C values for the Atyp.C-positive and -negative groups were 2.64 ± 0.69 and 0.38 ± 0.16, respectively. The optimal Atyp.C cutoff value determined by the receiver operating characteristic curve analysis was 0.4/µL. The area under the curve was 0.856, with a sensitivity of 79.5% and specificity of 85.1%. Atyp.C values of the UF-5000 showed high predictive performance for Atyp.C-positive specimens identified by urine sediment microscopy. d Conclusions: This study shows that a combination of UF-5000 analysis and microscopic examination of urine sediment improves Atyp.C detection in urine sediment analysis. These results suggest that Atyp.C measured by UF-5000 could be a useful screening parameter in routine testing of urine samples.

Novel absorbance peak of gentisic acid following the oxidation reaction.

Gentisic acid (GA), a metabolite of acetylsalicylic acid (ASA), and homogentisic acid (HGA), which is excreted at high levels in alkaptonuria, are divalent phenolic acids with very similar structures. Urine containing HGA is dark brown in color due to its oxidation. We recently reported a new oxidation method of HGA involving the addition of sodium hydroxide (NaOH) with sodium hypochlorite pentahydrate (NaOCl·5H2O), which is a strong oxidant. In the present study, we attempted to oxidize GA, which has a similar structure to HGA, using our method. We herein observed color changes in GA solution and analyzed the absorption spectra of GA after the addition of NaOH with NaOCl·5H2O. We also examined the oxidation reaction of GA using a liquid chromatography time-of-flight mass spectrometer (LC/TOF-MS). The results obtained indicated that GA solution had a unique absorption spectrum with a peak at approximately 500 nm through an oxidation reaction following the addition of NaOH with NaOCl·5H2O. This spectrophotometric method enables GA to be detected in sample solutions without expensive analytical instruments or a complex method.

Midstream urine sampling is necessary for accurate measurement of the urinary level of neutrophil gelatinase-associated lipocalin in healthy female subjects.

BACKGROUND: Urinary neutrophil gelatinase-associated lipocalin is an established biomarker of acute kidney injury, however, the levels are affected by the number of white blood cells in the urine. As we suspected the portion of the urinary stream sampled could also have a significant influence on the urinary NGAL levels in female subjects, we investigated the influence of the urine sampling procedure on the urinary NGAL levels. METHODS: We collected 25-mL urinary specimens from each of initial-stream and midstream urinary specimens, including 28 healthy adult female volunteers without kidney diseases or UTI. Then we compared the WBC count, NGAL level, and creatinine level between these specimens. RESULTS: We observed that the urinary NGAL levels were significantly higher in the specimens obtained from initial-stream urinary samples than in midstream specimens, and that they were strongly correlated with the leukocyte esterase activity and WBC count. Moreover, the differences in the urinary NGAL levels between the initial- and midstream urine samples were greater for initial-stream samples with higher leukocyte esterase activities, with a significant difference even for the initial-stream samples with no detectable leukocyte esterase activity. CONCLUSION: Therefore, midstream urine sampling is strongly recommended for accurate measurement of the urinary NGAL levels.

Absorbance measurements of oxidation of homogentisic acid accelerated by the addition of alkaline solution with sodium hypochlorite pentahydrate.

The urine of patients with alkaptonuria turns dark brown due to the oxidation of homogentisic acid (HGA) to benzoquinone acetic acid (BQA), and this is accelerated by the addition of alkali. We recently reported that alkaptonuric urine and HGA after the addition of alkali showed characteristic peaks at 406 and 430 nm. In order to improve the sensitivity of our spectrometric method for the detection of HGA, we accelerated the oxidation of HGA to BQA using sodium hypochlorite pentahydrate (NaOCl·5H2O), which is a strong oxidant. In the present study, we measured the absorption spectra of alkaptonuric urine and HGA solution after the addition of sodium hydroxide (NaOH) or NaOH with NaOCl·5H2O and analyzed the oxidation reaction of HGA after alkalization using a liquid chromatography time-of-flight mass spectrometer (LC/TOF-MS) and nuclear magnetic resonance (NMR) spectrometry. We accelerated the oxidation of HGA to BQA by adding NaOH with NaOCl·5H2O, and this absorbance measurement was useful for more sensitively observing the oxidation of HGA than LC/TOF-MS and NMR spectroscopy. This quick and easy screening method may be suitable for the diagnosis of alkaptonuria.

Enhancing the Detection of Dysmorphic Red Blood Cells and Renal Tubular Epithelial Cells with a Modified Urinalysis Protocol.

Urinary sediment is used to evaluate patients with possible urinary tract diseases. Currently, numerous protocols are applied to detect dysmorphic red blood cells (RBCs) and renal tubular epithelial cells (RTECs) in urinary sediment. However, distinct protocols are used by nephrologists and medical technologists for specimen concentration and observation, which leads to major discrepancies in the differential counts of formed elements such as dysmorphic RBCs and RTECs and might interfere with an accurate clinical diagnosis. To resolve these problems, we first tested a modified urinalysis protocol with an increased relative centrifuge force and concentration factor in 20 biopsy-confirmed glomerulonephritis patients with haematuria. We successfully improved the recovery ratio of dysmorphic RBCs in clinical specimens from 34.7% to 42.0% (P < 0.001). Furthermore, we confirmed the correlation between counts by the modified urinary protocol and Sysmex UF-1000i urinary flow cytometer (r ≥ 0.898, P < 0.001). A total of 28 types of isomorphic and dysmorphic RBCs were detected using a bright field microscope, with results comparable to those using a standard phase contrast microscope. Finally, we applied Sternheimer stain to enhance the contrast of RTECs in the urinary sediments. We concluded that this modified urinalysis protocol significantly enhanced the quality of urinalysis.

Novel round cells in urine sediment and their clinical implications.

BACKGROUND: Voided urine contains a variety of cells from the kidney and urinary tract and urinalysis provides us various information by investigating cellular components. We investigated urine sediment from renal impaired patients. RESULTS: We found 'round cell' to be distinct from known cells in sediment and is close to proximal convoluted tubule-derived cells based on morphology and molecular marker expression (GGT1 but not podocalyxin). Also it was positive for undifferentiated cell markers, including PAX2, WT1, OSR1, and SIX2. They were observed in end-stage renal failure patients and the number of cells was correlated with the severity of chronic kidney disease. A prospective analysis revealed that patients who had more round cells were more likely to require hemodialysis within a year. CONCLUSION: Round cells are a novel marker that can be used to predict the need for hemodialysis.

Detection of novel visible-light region absorbance peaks in the urine after alkalization in patients with alkaptonuria.

BACKGROUND: Alkaptonuria, caused by a deficiency of homogentisate 1,2-dioxygenase, results in the accumulation of homogentisic acid (2,5-dihydroxyphenylacetic acid, HGA) in the urine. Alkaptonuria is suspected when the urine changes color after it is left to stand at room temperature for several hours to days; oxidation of homogentisic acid to benzoquinone acetic acid underlies this color change, which is accelerated by the addition of alkali. In an attempt to develop a facile screening test for alkaptonuria, we added alkali to urine samples obtained from patients with alkaptonuria and measured the absorbance spectra in the visible light region. METHODS: We evaluated the characteristics of the absorption spectra of urine samples obtained from patients with alkaptonuria (n = 2) and compared them with those of urine specimens obtained from healthy volunteers (n = 5) and patients with phenylketonuria (n = 3), and also of synthetic homogentisic acid solution after alkalization. Alkalization of the urine samples and HGA solution was carried out by the addition of NaOH, KOH or NH4OH. The sample solutions were incubated at room temperature for 1 min, followed by measurement of the absorption spectra. RESULTS: Addition of alkali to alkaptonuric urine yielded characteristic absorption peaks at 406 nm and 430 nm; an identical result was obtained from HGA solution after alkalization. The absorbance values at both 406 nm and 430 nm increased in a time-dependent manner. In addition, the absorbance values at these peaks were greater in strongly alkaline samples (NaOH- KOH-added) as compared with those in weakly alkaline samples (NH4OH-added). In addition, the peaks disappeared following the addition of ascorbic acid to the samples. CONCLUSIONS: We found two characteristic peaks at 406 nm and 430 nm in both alkaptonuric urine and HGA solution after alkalization. This new quick and easy method may pave the way for the development of an easy method for the diagnosis of alkaptonuria.

[How Far We can Diagnose by Urine Sediment Tests?].

Morphological examinations for renal disease are mainly renal biopsy and urine sediment tests. Biopsy specimens are now evaluated in detail, and test items are evaluated as highly sensitive and specific compared with urine sediments, which reflect renal changes indirectly and are low in sensitivity and specificity. On the other hand, the standardization of urine sediment tests is now in progress, and some labs can provide sediment results beyond screening for distracted RBC, differential WBC, or atypical cells. The diagnostic importance of sediments is revisited.

[Chairmen's introductory remarks].

Recently, mild to moderate renal dysfunction has attracted attention as a new common disease in Japan as well as all over the world. Also, the morbidity of endstage renal disease and progression to dialysis is increasing each year. It is now widely recognized that renal dysfunction is a major risk for cardiovascular disease; therefore, cardio-renal relationships should be investigated. To elucidate the pathophysiology of this relationship and develop new methods for early diagnosis, treatment and prevention can promise a long and healthy life. It is also important to provide high quality data on urinalysis and kidney function.