Micral-test strips evaluated for screening for albuminuria.

We have evaluated Micral-Test, an immunochemical strip test specific for albumin, as a screening tool for slight ("micro") albuminuria. First morning urine samples containing albumin concentrations (by radioimmunoassay) of 0.4-440 mg/L were collected from 112 diabetic patients. The Micral-Test result for each sample was assessed by one observer. All 34 samples having albumin concentrations greater than or equal to 20 mg/L and 71 of 78 samples less than 20 mg/L were correctly identified, giving 100% sensitivity and 91% specificity. Six samples were measured 10 times by one observer: three samples were read consistently; one, albumin concentration 86 mg/L, was read as 50 and 100 mg/L; and two, albumin concentrations 32 and 38 mg/L, were read as 20 and 50 mg/L, respectively. Contact with urine for 2 s rather than the recommended 5 s resulted in an underestimation of the albumin concentration in 13 of 35 samples (Z = -3.18, P = 0.001), as did taking readings earlier than the recommended 5 min (Z = -3.92, P less than 0.001). Six observers independently performed Micral-Test measurements on 10 samples. Eight samples were correctly classified as greater than or equal to 20 or less than 20 mg/L by all observers, but two (albumin concentrations 25 and 18 mg/L) were misclassified by at least one observer. The Micral-Test is a sensitive and specific screening tool, but is semiquantitative and critically time dependent.     

Comparison of four commercial urinary albumin (microalbumin) methods: implications for detecting diabetic nephropathy using random urine specimens

The results of four urinary albumin methods used to identify patients with early diabetic renal disease were compared using random urine samples from healthy and diabetic patients. These methods were the Beckman Array and Behring BNAI immunonephelometric methods, the Dade aca particle-enhanced turbidimetric inhibition immunoassay method, and the INCSTAR SPQ immunoturbidimetric method. The albumin/creatinine ratio reference interval was found to be 2–20 mg albumin/g creatinine (mg/g) for the Array and 3.5–27.5 mg/g for the aca method. All four methods were compared using urines from a group of diabetic and nondiabetic patients. The BNAI, SPQ and Array methods compared well with one another while the aca demonstrated a positive bias of almost 60% at the 30 mg/g and 300 mg/g levels with certain lots of reagent and calibrator. Calibrator cross-over experiments demonstrated that some of the positive bias of the aca method could be accounted for by calibrator differences.     

Immunoturbidimetry of urinary albumin: prevention of adsorption of albumin; influence of other urinary constituents

I describe a simple, rapid immunoturbidimetric assay for low concentrations of albumin in urine (2 to 260 mg/L). However, in this assay, the human serum albumin (HSA) in the standards binds nonspecifically to the polystyrene or glass tubes. This nonspecific binding cannot be prevented by adding bovine serum albumin (BSA) to standards, because the anti-HSA antibody cross reacts with BSA. Adding Triton X-100 (1 mL/L) to standards effectively prevents this nonspecific binding of HSA from standards to both polystyrene and glass tubes. High concentrations of compounds found in urine from normal and diabetic subjects do not interfere with this assay if pH extremes can be avoided. The between-day CV is 4.8% at means = 18.8 mg/L and 2.0% at means = 183.1 mg/L. Measurements by this immunoturbidimetric method (y) correlate well with those obtained by a radioimmunoassay (x): y = 1.078x - 0.141 mg/L (n = 98; r = 0.984) and with those obtained by a radial immunodiffusion method (x'): y = 1.026x' - 0.117 mg/L (n = 98; r = 0.983). Urinary excretion of albumin by 25 healthy, nondiabetic subjects was less than 8 micrograms/min.     

Increased urinary haemoglobin in diabetics with microalbuminuria--measured by an ELISA

A solid-phase sandwich enzyme immunoassay for the determination of urinary haemoglobin is described. A screening study was performed to establish whether occult haematuria/haemoglobinuria is present in diabetic patients with elevated urinary albumin excretion. Non-insulin-dependent diabetic patients (145) aged 66.5 years +/- 5.6 with a known duration of diabetes of 10.4 years +/- 6.8 were studied. They delivered a first morning urine sample at consecutive outpatient visits. Erythrocytes were lysed by freezing of the urine samples. The patients were categorized into three groups according to urinary albumin concentration (UAC) as measured by radio-immunoassay. Forty-five patients with microalbuminuria i.e. UAC greater than 20- less than or equal to 200 micrograms/ml had an elevated urinary haemoglobin concentration (UHC) of 38.5 micrograms/l compared to 90 patients with normal UAC and a UHC of 7.7 micrograms/l, p less than 10(-4). A further increase was seen in 10 proteinuric patients, UHC 161.8 micrograms/l, p less than 0.05. The urinary concentration of albumin and haemoglobin were significantly correlated, r = 0.49, p less than 10(-8). In 32 insulin-dependent diabetics the findings were similar. There was no correlation between either age or known duration and the haemoglobin concentration. In 42 normal subjects, the UHC was 3.6 micrograms/lx/divided by 6.1. Haematuria has formerly been described in patients with diabetic nephropathy alone. The present findings suggest that occult haematuria/haemoglobinuria is already present in patients with microalbuminuria.     

Immunoturbidimetry: an attractive technique for the determination of urinary albumin and transferrin

Immunoturbidimetry is a suitable technique for the determination of urinary proteins in clinical laboratories. We have developed immunoturbidimetric assays for the measurement of urinary albumin and transferrin which are adapted for the Cobas-BIO centrifugal analyzer. Linear range for the albumin assay was 6.25-167 mg/L and for the transferrin assay 1.66-6.66 mg/L. The analytical recoveries of albumin and transferrin averaged 96%. The addition of up to 50 g/L hemoglobin to urine did not interfere with the determination of either protein. Within-run and between-run imprecision for albumin assay was 2.2% and 3.2% at an albumin level of 140 mg/L, and 4.8% and 8.7% at an albumin level of 16 mg/L; for transferrin assay the respective imprecisions were 3.6% and 6.7% at transferrin concentration of 6 mg/L, and 5.2% and 11.1% at a transferrin concentration of 1.5 mg/L. Reference ranges for both assays were established using urines from 17 healthy individuals; albumin had a range of 0-22 mg/L and transferrin 0-1.9 mg/L.     

Albumin excretion rate, albumin concentration, and albumin/creatinine ratio compared for screening diabetics for slight albuminuria

Slight albuminuria, an overnight albumin excretion rate (AER) greater than 30 micrograms/min in an "Albustix"-negative sample, predicts development of diabetic nephropathy. This study compares the AERs for 261 timed overnight urine collections with the albumin concentrations and albumin/creatinine ratios for the same specimens (equivalent to first morning specimens). Thirty-one specimens (11.9%) had AERs greater than 30 micrograms/min. Use of an albumin/creatinine ratio greater than 3.0 mg/mmol to predict an AER greater than 30 micrograms/min gave a sensitivity of 96.8%, a specificity of 93.9%, and a predictive value of 68.2%, with a correlation coefficient of 0.921. Use of an albumin concentration greater than 17 mg/L gave a sensitivity of 96.8%, a specificity of 90.9%, a predictive value of 58.8%, and a slightly poorer correlation (r = 0.904). Evidently either method is acceptable as an initial screening procedure, but determination of albumin concentration alone would be preferable because of lesser cost.     

不同时段尿白蛋白在诊断早期糖尿病肾脏损伤中的应用

目的 研究糖尿病患者不同时段尿白蛋白(urinary albumin)的排泌情况及尿白蛋白在诊断早期糖尿病肾脏损伤中的应用.方法 收集中山医院门诊及住院糖尿病患者及健康对照组3 d内不同时间段的尿液,分析尿白蛋白天内、天间的排泌变化情况;以24 h尿白蛋白为标准判断肾脏早期损伤情况,比较不同时段尿及时间点尿与24 h尿白蛋白的相关性、诊断特异度及敏感度;评估随机尿的诊断特异度及敏感度,推导随机尿最佳诊断水平.结果尿白蛋白天间变异较大,以尿Cr和尿量分别校正后可降低变异.糖尿病组中尿白蛋白使用尿Cr校正后变异系数(CV)小于尿量校正(CV分别为49%±23%vs 64%±30%).尿白蛋白天内排泌呈节律性变化.不同尿液留取方式中夜间尿尿白蛋白/尿Cr(ratio of urinary concentrations of albumin and creatinine,ACR)与24 h尿白蛋白定量相关性最好(R~2=0.976),优于晨尿ACR(R~2=0.900)、午间餐后尿ACR(R~2=0.584)和随机尿ACR(R2=0.791).以24 h尿白蛋白总量作为判断标准进行受试者操作特性曲线(ROC曲线)分析显示,随机尿ACR的判断值为27.7 μg/mg尿Cr(存在男女性别差异:男性12.8μg/mg尿Cr vs性27.0μg/mg尿Cr).最小阴性似然比0.011时推导随机尿ACR的排除判断值为13.0 μg/mg尿Cr;最大阳性似然比481.000时推导随机尿ACR的确诊判断值为87.4 μg/mg尿Cr.结论 尿Cr较尿量能更好地降低尿白蛋白天内变异,但仍无法完全消除变异.夜尿ACR与24 h尿白蛋白定量相关性最好,可替代24 h尿白蛋白定量.随机尿ACR作为最方便留取的尿液标本亦可以较好地替代24 h尿白蛋白定量,但应考虑引入尿Cr后带来的性别间差异.以13.0 μg/mg及87.4 μg/mg作为随机尿ACR的排除判断值及确诊判断值可以便于临床医师基本排除或确定白蛋白尿的出现.     

Presence of immunounreactive albumin in the urine of diabetic patients

Recent studies have demonstrated that conventional immunochemical assays underestimate urinary albumin concentration because of the presence of immunounreactive albumin. It has been reported that intact urinary albumin in 24-hr diabetic urine samples could be detected as total concentration (immunoreactive+immunounreactive) by an HPLC method based on size exclusion chromatography. The aim of this study was to investigate urinary albumin concentration in diabetic spot urine samples by comparing the HPLC method with several other methods. The albumin concentrations on 80 diabetic spot urine specimens were measured by turbidimetric immunoassay (TIA), high performance liquid chromatography (HPLC), and a dipstick method.In addition, they were also analyzed by reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and native polyacrylamide gel electrophoresis (Native PAGE). The albumin concentrations derived from diabetic spot urine samples measured by the HPLC method were higher than those of the other methods except for five of 80 samples. Furthermore, the albumin concentrations analyzed by Native PAGE were higher than SDS PAGE in 61 (76.2%) of 80 samples. This study suggests the need for evaluating diabetes not only by HPLC, but also by combining it with another method.     

Analytical validation of an HPLC assay for urinary albumin

BACKGROUND: Microalbuminuria is the earliest clinical finding for renal disease. Diabetic individuals often produce modified forms of albumin, perhaps due to impaired lysosomal processing, that are undetectable by common immunoassays but accurately measured by HPLC. METHODS: We evaluated the performance of a commercially available, FDA-approved HPLC assay (AusAm Biotechnologies, NY) and compare results to our immunoturbidimetric assay (ITA, Beckman-Coulter, CA) using random urine specimens from 32 nondiabetic and 60 type 1 and 2 diabetic subjects. RESULTS: The HPLC assay was linear to 963 mg/l with a limit of detection of 6.1 mg/l. Within-run and between-run precision was <2% and 7-10%, respectively. Unpreserved urine was stable for at least 3 days at room temperature and 10 days at 4 degrees C. In both diabetic and nondiabetic subjects urinary albumin concentrations were higher by HPLC than by ITA, and many more diabetic and nondiabetic individuals were classified as microalbuminuric by HPLC than by ITA. The HPLC assay showed acceptable performance; however, because urinary albumin concentrations are higher in apparently healthy nondiabetic as well as diabetic subjects, different cutpoints will be necessary to accurately differentiate microalbuminuria. CONCLUSIONS: Prospective studies are necessary to determine whether the HPLC assay can effectively detect microalbuminuria earlier than current assays without a concomitant increase in the false positive rate.     

不同时段尿微量清蛋白对糖尿病早期肾损害的诊断价值

目的探讨2型糖尿病肾病患者不同时段尿微量清蛋白(u-MA)变化及其对2型糖尿病患者早期肾损伤的诊断价值。方法收集糖尿病肾病患者组(n=27)和健康对照组(n=30)连续3 d的晨尿、餐后尿、随机尿和24 h尿,进行尿微量清蛋白测定。结果糖尿病患者尿微量清蛋白排泌高峰为餐后尿(61.7±26.5 mg/L),其次为晨尿(58.9±23.5 mg/L)和24 h尿(56.6±13.2 mg/L),3者显著高于随机尿(37.2±21.4 mg/L)(P<0.05),且各时段尿微量清蛋白都有较大的日间差异,以随机尿差异最大CV=57.5%,餐后尿、晨尿、和24 h尿分别为42.9%、39.9%和23.3%,糖尿病肾病患者各时段尿微量清蛋白与对照组比较差异有统计学意义(P<0.05)。结论餐后尿和晨尿微量清蛋白含量的多次检测有利于提高2型糖尿病患者早期肾损害的诊断。     

2型糖尿病患者微量白蛋白尿临界值的研究

目的研究2型糖尿病(T2DM)患者晨尿、随机尿的尿白蛋白/肌酐比值(ACR)及尿白蛋白(U-Alb)浓度,判定微量白蛋白尿的临界值,以检测早期糖尿病肾脏疾病(DKD)。方法收集169例T2DM患者及40名健康体检者(正常对照组)的24 h尿、晨尿、随机尿,以24 h尿白蛋白排泄率(UAE)为早期DKD的判定标准,分析目前临床应用的晨尿、随机尿的ACR及U-Alb浓度判定微量白蛋白尿的临界值对早期DKD的筛查效能;根据受试者工作特征(ROC)曲线分析,Youden指数最大时对应的晨尿、随机尿的ACR及U-Alb浓度为检测早期DKD微量白蛋白尿的临界值。结果在微量白蛋白尿判定结果中,晨尿ACR与24 h UAE的符合率为43%,UAlb为37%;随机尿ACR为48%,U-Alb为41%,以上4个指标与24 h UAE的判定结果有明显差异(P均0.001)。正常对照组晨尿、随机尿检测结果判定与24 h UAE一致,均为正常。ROC曲线分析显示ACR晨尿临界值为男16 mg/g、女23 mg/g(Youden指数分别为0.70、0.67,阴性预测值分别为97%、100%,阳性预测值分别为72%、65%);ACR随机尿临界值为男17 mg/g、女28 mg/g(Youden指数分别为0.68、0.67,阴性预测值分别为90%、90%,阳性预测值分别为61%、82%);U-Alb晨尿临界值为男16 mg/L、女15 mg/L(Youden指数分别为0.57、0.59,阴性预测值分别为84%、87%,阳性预测值分别为90%、73%);U-Alb随机尿临界值为男17 mg/L、女14 mg/L(Youden指数分别为0.56、0.53,阴性预测值分别为73%、81%,阳性预测值分别为85%、71%)。ACR的最大Youden指数均0.6,且高于相应的U-Alb浓度的Youden指数。结论目前临床应用的晨尿、随机尿微量白蛋白尿的临界值判定T2DM患者早期肾病的漏诊率较高,应重新建立T2DM患者晨尿、随机尿ACR及U-Alb的临界值,以利于DKD的早期防治。     

Screening for microalbuminuria in patients with diabetes mellitus: frozen storage of urine samples decreases their albumin content

The influence of storage on urinary albumin concentration was prospectively studied with use of overnight urine specimens (Albustix negative) from 73 diabetic patients. From each urine sample four aliquots were taken. One was stored at 4 degrees C and assayed within two weeks, the other three were stored at -20 degrees C and assayed within two weeks and after two and six months. Albumin concentration was measured with laser immunonephelometry. The detection limit, 1 mg/L, suffices for the screening of diabetic patients for microalbuminuria. After storage for two and six months at -20 degrees C, significantly lower albumin concentrations were found. The difference was mainly caused by lower concentrations found in urine samples in which a precipitate had formed, which was the case in 22 and 25 samples, respectively. Thus, freezing of urine samples for determination of low concentrations of albumin may yield falsely low results. Urine samples are best stored at 4 degrees C and assayed within two weeks.     

Enzyme immunoassay: an improved determination of urinary albumin in diabetics with incipient nephropathy

An enzyme linked immunoadsorbent assay for urinary albumin using commercially available reagents is described. The assay range is 2.5-120 micrograms/l. When samples are analysed in two standard dilutions, the assayable albumin concentration range is 2.5-240 mg/l, covering the clinical range from normoalbuminuria to overt clinical nephropathy. Intra-assay variation was 2.1% and interassay variation 8.3%. Recovery of added albumin to urine was 95%-106% and dilution of urine was linear. The correlation to urinary albumin determined by immunodiffusion was excellent (n = 80, r = 0.99). Intraindividual variation of the 24 h urine albumin excretion of different days was high in patients with incipient diabetic nephropathy (51.5%) and was only slightly reduced by taking the variation of creatinine excretion into account (39.5%). No correlation was found between albumin excretion, and HbA1c or urine glucose excretion, indicating that minor metabolic variations are not responsible for the huge intraindividual day-to-day variations of UalbV. The study shows that more than one UalbV measurement must be done before classifying patients into groups with or without incipient diabetic nephropathy.     

Assessment of immunochemical methods for determining low concentrations of albumin in urine

Four immunochemical methods (radioimmunoassay, RIA; radial immunodiffusion, RID; immunoturbidimetry, IT; enzyme-linked immunosorbent assay, ELISA) for measuring urinary albumin at low concentrations were assessed for their assay characteristics and practicability. Precision and accuracy were comparable between the methods when studied individually. We made a method comparison, with RIA as reference, using urine samples from diabetic patients with albumin concentrations ranging from 1 to 120 mg/L. There was no significant systematic difference between RID and RIA, but IT and ELISA gave consistently lower values than RIA, the mean differences being 1.8 (p less than 0.01) and 9.7 mg/L (p less than 0.001), respectively. Random error, compared with that for RIA, was in increasing order: RID (residual SD = 3.8 mg/L); IT (4.3 mg/L); ELISA (7.3 mg/L). The difference between the methods increased with the albumin concentration. Operational cost was highest with IT, lowest with RIA. Capital cost was highest with RIA and lowest with RID, which required most technical skill. ELISA had intermediate overall costs.     

Immunoturbidimetric assay for the determination of microalbuminuria using the Hitachi analyser

A sensitive and specific immunoturbidimetric method is described for the determination of low concentrations of urinary albumin using a Hitachi 704 or 705 analyser. The sensitivity was 1 mg/l and the precision attained was good (CV 15%, 7% and 6% for low, medium and higher albumin concentrations). The assay was used to determine urinary albumin excretion rates in healthy controls (less than 15 micrograms/min) and in type I diabetics. Since microalbuminuria (30-200 micrograms/min) seems to be a good predictor for the development of diabetic nephropathy and other late diabetic complications, this assay is suitable for the necessary screening and follow-up of diabetic nephropathy. In contrast to RIA methods, no radioactive tracers are needed.     

Microalbuminuria. Invalidity of simple concentration-based screening tests for early nephropathy due to urinary volumes of diabetic patients

OBJECTIVE: To evaluate the possibility of replacing quantitative albumin excretion rate (AER) measurements with rapid screening tests for microalbuminuria. RESEARCH DESIGN AND METHODS: Dipstick-negative specimens from 363 consecutive insulin-dependent diabetes mellitus (IDDM) and 46 non-insulin-dependent diabetes mellitus (NIDDM) patients from primary-care and hospital clinics (11% inpatients) within the district of Turku University Hospital were studied. Albumin concentrations and 12-h nightly excretion rates (N-AER) were measured by nephelometry (sensitivity 2 mg/L). RESULTS: An increased N-AER (greater than 15 micrograms/min) was seen in 99 IDDM (27%) and 15 NIDDM (33%) patients. The median urinary volume was 900 ml/12 h, with a maximum of 3000 ml. At the level of 20 mg albumin/L, the sensitivity to detect elevated N-AER was 70% among IDDM patients and 60% among NIDDM patients. At a lower albumin concentration of 10 mg/L, the sensitivities were increased to 91 and 87% in IDDM and NIDDM patients, respectively, but the specificities were reduced to 77 and 71%, respectively. CONCLUSIONS: To evaluate incipient nephropathy, we recommend quantitative measurements of N-AER from timed urine collections only. Dipstick tests are either insensitive or nonspecific.     

Evaluation of an immunoturbidimetric microalbuminuria assay

We have evaluated an immunoturbidimetric method for the estimation of urinary albumin. The method, besides being easy to perform and cost-effective, was sensitive enough to detect an even slightly increased albumin excretion (detection limit 5 mg/l). Within-run reproducibility was 1.8 and 2.1%, and between-run reproducibility 2.9 and 4.3% in samples containing 16.1-17.8 mg/l and 50.6-54.0 mg/l of albumin, respectively. The recovery of albumin added to the samples was 98.6-106.6%. Results obtained by this method correlated well with the results obtained by radial immunodiffusion (r = 0.980, n = 44) and radioimmunoassay (r = 0.982, n = 41). The immunoturbidimetric method can be easily adapted for several clinical chemistry analysers.     

Detection of microalbuminuria in diabetic patients using a simple latex agglutination test

The adaptation of an immunoturbidimetric assay to quantitatively measure urinary albumin is described and this was used to evaluate a commercial latex agglutination slide test, Albuscreen, with a sensitivity of 25 mg/l. Excellent agreement was observed between the two methods, and in clinical studies an acceptable classification of patients was made using Albuscreen, an albumin: creatinine ratio of less than or equal to 2.5 being used to indicate possible microalbuminuria. A small number of false negative and false positive results occurred in some dilute and concentrated urine samples, respectively.     

糖尿病患者尿微量清蛋白、尿β_2-微球蛋白、糖化血红蛋白及血脂联合检测的临床意义

目的分析糖尿病患者的尿微量清蛋白、β2-微球蛋白、糖化血红蛋白及血脂含量,以观察其与糖尿病肾脏微血管病变的相关性。方法糖尿病患者根据尿微量清蛋白(mALb)分为两组:(1)糖尿病肾病组,晨尿高微量清蛋白(20mg/L)30例;(2)无糖尿病肾病组,晨尿正常微量清蛋白(20mg/L))33例。结果晨尿高微量清蛋白组的尿β2-微球蛋白、糖化血红蛋白及胆固醇含量显著高于晨尿正常微量清蛋白组,两组间差异有统计学意义。结论尿微量清蛋白(mALb)及尿β2-微球蛋白是糖尿病早期肾小球及肾小管损伤的标志物,肾病微血管病变程度与HbA1c的增高有关,血脂异常可增加糖尿病患者肾病微血管病变的危险性。联合检测对糖尿病肾病的早期预防诊断及治疗有重要的临床意义。     

Urinary albumin excretion in normal subjects and in diabetic patients measured by a radioimmunoassay: methodological and clinical aspects

We have developed a radioimmunoassay method (RIA) to measure urinary albumin excretion. We determined the albumin excretion rate (AER) (micrograms/min) of 122 healthy subjects and 145 diabetic patients (115 type I, 30 type II). The results indicate that the RIA is sensitive (0.39 +/- 0.08 mg/L), precise (CV 5-8%), and gives reliable results on previously frozen urine samples. The distribution of the AER values in healthy subjects and diabetic patients was not normal. It was normalized by log or square-root transformation of the data. Seventy-three percent of diabetic patients lay within the normal range (0.6-10.6 micrograms/min). Twenty percent could be considered "at risk" to develop overt diabetic nephropathy because their albuminuria exceeded a threshold level of 15 micrograms/min chosen previously as the cutoff value for microalbuminuria. We found no correlation between AER and glycated hemoglobin, and only a weak correlation between AER and diabetes duration in type I diabetic patients.