Seegmiller, Jesse C.

Abstract Background Iohexol is used for measurement of kidney glomerular filtration rate (GFR). Until recently, there have not been available proficiency standards to assist in calibrating a laboratory's results. In view of a shift in calibration at the University of Rochester Medical Center (URMC) laboratory, serving as Central Biochemistry Laboratory for the CKiD study, we performed a multicentered laboratory comparison. Methods Two batches of 30 fortified sera and patient samples from serum or heparinized plasma were sent for duplicate analysis to URMC, University of Minnesota (UMN), Mayo Clinic, and University of Lund. Five proficiency testing materials from Equalis AB were also provided. Iohexol calibration was performed using dilutions of OmnipaqueTM 300 and concentrations measured by HPLC or LC-MS/MS (Mayo). Results The 2 batches sent to UMN and University of Lund agreed well. URMC calibration was 11%–13% lower, and Mayo was 4%–8% lower for fortified samples. URMC corrected calibration was 3%–8% higher for these samples. When measured values were adjusted for the results of the Equalis samples, all laboratories agreed within 1%–2% on all iohexol concentrations. Conclusions For 12 URMC calibrator lots from November 2006 to March 2016, the factor quantifying the underestimation of measured to true iohexol concentration was 0.89. If each concentration was divided by 0.89, the calculated GFRs would be reduced by 10%–11%. GFR results for CKiD were adjusted for this shift in calibration. Regular examination of iohexol proficiency testing materials, free exchange of samples among laboratories, and standardized dilution of the stock iohexol for calibration would help to bring more universal agreement to this assay.

Abstract Background Urine albumin is a key laboratory test used for classification, assessment of risk, and monitoring treatment of patients with chronic kidney disease. Urine albumin measurement results are not standardized among different measurement procedures. Consequently, clinical guidelines using fixed decision values for urine albumin cannot be applied consistently. Content Isotope dilution mass spectrometry reference measurement procedures and certified reference materials are being developed to enable standardization of immunoassay measurement procedures for urine albumin. A previous report determined calibration bias was the major error source for differences in results among different measurement procedures for urine albumin. Performance goals for between-day precision, ≤6% CV above 15 mg/L, and for specimen-specific effects, ≤6% CV, were established on the basis of the performance capability of current measurement procedures. The biological variation model was used to estimate a total allowable error of ≤24%–30% and from that the goal for bias of ≤7%–13%. Summary A reference system of higher-order certified reference materials and reference measurement procedures is being developed to enable standardization of urine albumin measurement results. Goals have been established for total allowable error, specimen-specific effects, imprecision, and bias to facilitate efforts to standardize urine albumin measurement results.

Abstract Background: Glomerular filtration rate (GFR) can be determined by measuring renal clearance of the radiocontrast agent iothalamate. Current analytic methods for quantifying iothalamate concentrations in plasma and urine using liquid chromatography or capillary electrophoresis have limitations such as long analysis times and susceptibility to interferences. We developed a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method to overcome these limitations. Methods: Urine and plasma samples were deproteinized using acetonitrile and centrifugation. The supernatant was diluted in water and analyzed by LC-MS/MS using a water:methanol gradient. We monitored 4 multiple reaction monitoring transitions: m/z 614.8–487.0, 614.8–456.0, 614.8–361.1, and 614.8–177.1. We compared the results to those obtained via our standard capillary electrophoresis (CE-UV) on samples from 53 patients undergoing clinical GFR testing. Results: Mean recovery was 90%–110% in both urine and plasma matrices. Imprecision was ≤15% for the m/z 614.8–487.0 and 614.8–456.0 transitions over a 10-day period at 1 mg/L. Method comparison for 159 patient samples (53 clearances) provided the following Passing–Bablok regressions: plasma iothalamate LC-MS/MS (y) vs CE-UV (x), y = 0.99x + 0.36; urine iothalamate LC-MS/MS vs CE-UV, y = 1.01x + 0.31; corrected GFR LC-MS/MS vs CE-UV, y = 1.00x + 0.00. Interfering substances prevented accurate iothalamate quantification by CE-UV in 2 patients, whereas these samples could be analyzed by LC-MS/MS. Conclusions: Iothalamate can be quantified by LC-MS/MS for GFR measurement. This method circumvents potential problems with interfering substances that occasionally confound accurate GFR determinations.

Abstract Background: Increased urinary albumin excretion is a well-documented diagnostic and prognostic biomarker for renal disease. Urinary albumin is typically measured in clinical settings by immunoassay methods. However, neither a reference method nor a urine albumin calibration reference material is currently available. Methods: We quantified urinary albumin in patient samples by using 3 commercially available reagent systems: DiaSorin SPQ™ and Beckman Coulter LX® 20 (immunoturbidimetric), and Siemens Immulite® (competitive immunoassay). Results were compared to values obtained by protein-cleavage liquid chromatography–tandem mass spectrometry (LC-MS/MS). Results: In general, results from the 3 immunoassays agreed with results from LC-MS/MS. However, the SPQ results showed a negative bias across all ranges of albuminuria [(0–200 mg/L, y = 0.91x – 3.74 (CI 0.86–0.96); > 200 mg/L, y = 0.88x – 40.30 (CI 0.76–1.00)], whereas the LX 20 showed minimal bias in the 0–200 mg/L range [y = 0.97x − 88 (CI 0.92–1.02)] and the Immulite assay showed positive bias in the 0–200 mg/L range [y = 1.15x – 4.38 (CI 1.09–1.20)]. Conclusions: These results showed a reasonable quantification of urinary albumin by representative polyclonal and monoclonal immunoassays compared to an LC-MS/MS assay. In addition, the results do not suggest the presence of nonimmunoreactive albumin in urine. However, differences in analytic performance between assays support the need for a reference calibration material and reference method to standardize clinical laboratory measurements of urinary albumin.

Abstract Background: Urinary albumin excretion is a sensitive diagnostic and prognostic marker for renal disease. Therefore, measurement of urinary albumin must be accurate and precise. We have developed a method to quantify intact urinary albumin with a low limit of quantification (LOQ). Methods: We constructed an external calibration curve using purified human serum albumin (HSA) added to a charcoal-stripped urine matrix. We then added an internal standard, 15N-labeled recombinant HSA (15NrHSA), to the calibrators, controls, and patient urine samples. The samples were reduced, alkylated, and digested with trypsin. The concentration of albumin in each sample was determined by liquid chromatography–tandem mass spectrometry (LC-MS/MS) and linear regression analysis, in which the relative abundance area ratio of the tryptic peptides 42LVNEVTEFAK51 and 526QTALVELVK534 from albumin and 15NrHSA were referenced to the calibration curve. Results: The lower limit of quantification was 3.13 mg/L, and the linear dynamic range was 3.13–200 mg/L. Replicate digests from low, medium, and high controls (n = 5) gave intraassay imprecision CVs of 2.8%–11.0% for the peptide 42LVNEVTEFAK51, and 1.9%–12.3% for the 526QTALVELVK534 peptide. Interassay imprecision of the controls for a period of 10 consecutive days (n = 10) yielded CVs of 1.5%–14.8% for the 42LVNEVTEFAK51 peptide, and 6.4%–14.1% for the 526QTALVELVK534 peptide. For the 42LVNEVTEFAK51 peptide, a method comparison between LC-MS/MS and an immunoturbidometric method for 138 patient samples gave an R2 value of 0.97 and a regression line of y = 0.99x + 23.16. Conclusions: Urinary albumin can be quantified by a protein cleavage LC-MS/MS method using a 15NrHSA internal standard. This method provides improved analytical performance in the clinically relevant range compared to a commercially available immunoturbidometric assay.

Abstract Background: The accurate and precise measurement of urinary albumin is critical, since even minor increases are diagnostically sensitive indicators of renal disease, cardiovascular events, and risk for death. To gain insights into potential measurement biases, we systematically compared urine albumin measurements performed by LC-MS, a clinically available immunoturbidimetric assay, and size-exclusion HPLC. Methods: We obtained unused clinical urine samples from 150 patients who were stratified by degrees of albuminuria (<20 mg/L, 20–250 mg/L, >250 mg/L) as determined by the immunoturbidimetric assay used in our clinical laboratory (Roche Hitachi 912). Urine albumin was then remeasured via LC-MS and HPLC (Accumin™) assays. Results: The immunoturbidimetric assay, calibrated using manufacturer-supplied serum-derived calibrators (Diasorin), underestimated albumin compared with LC-MS. After calibration with purified HSA, this immunoturbidimetric assay correlated well with LC-MS. HPLC overestimated albumin compared with both LC-MS and immunoturbidimetry. The current LC-MS and HPLC assays both performed poorly at concentrations <20 mg/L. Conclusions: Efforts are needed to establish gold-standard traceable calibrators for clinical assays. LC-MS is a specific method to quantify albumin in native urine when concentrations exceed 20 mg/L, and therefore could be employed for standardization among assays.