Long-Term Longitudinal Stability of Kidney Filtration Marker Measurements: Implications for Epidemiological Studies and Clinical Care.

BACKGROUND: Establishment and improvement of glomerular filtration rate estimating equations requires accurate and precise laboratory measurement procedures (MPs) for filtration markers. The Advanced Research and Diagnostic Laboratory (ARDL) at the University of Minnesota, which has served as the central laboratory for the Chronic Kidney Disease Epidemiology Collaboration since 2009, has implemented several quality assurance measures to monitor the accuracy and stability of filtration marker assays over time. METHODS: To assess longitudinal stability for filtration marker assays, a 40-sample calibration panel was created using pooled serum, divided into multiple frozen aliquots stored at -80 °C. ARDL monitored 4 markers-creatinine, cystatin C, beta-2-microglobulin (B2M) and beta-trace protein-measuring 15 calibration panel aliquots from 2009 to 2019. Initial target values were established using the mean of the first 3 measurements performed in 2009-10, and differences from target were monitored over time. New MPs for cystatin C and B2M were added in 2012, with target values established using the first measurement. RESULTS: The mean percentage difference from mean target values across time was <2% for all original MPs (-0.59% for creatinine; -0.94% for cystatin C; -0.82% for B2M; 1.24% for beta-trace protein). CONCLUSIONS: Close monitoring of filtration marker trends with a calibration panel at ARDL demonstrates remarkable long-term stability of the MPs. Routine use of a calibration panel for both research studies and clinical care is recommended for filtration markers where longitudinal monitoring is important to detect analytical biases, which can mask or confound true clinical trends in patients.

Performance in Measurement of Serum Cystatin C by Laboratories Participating in the College of American Pathologists 2014 CYS Survey.

CONTEXT: Cystatin C is becoming an increasingly popular biomarker for estimating glomerular filtration rate, and accurate measurements of cystatin C concentrations are necessary for accurate estimates of glomerular filtration rate. OBJECTIVE: To assess the accuracy of cystatin C concentration measurements in laboratories participating in the College of American Pathologists CYS Survey. DESIGN: Two fresh frozen serum pools, the first from apparently healthy donors and the second from patients with chronic kidney disease, were prepared and distributed to laboratories participating in the CYS Survey along with the 2 usual processed human plasma samples. Target values were established for each pool by using 2 immunoassays and ERM DA471/IFCC international reference material. RESULTS: For the normal fresh frozen pool (ERM-DA471/IFCC-traceable target of 0.960 mg/L), the all-method mean (SD, % coefficient of variation [CV]) reported by all of the 123 reporting laboratories was 0.894 mg/L (0.128 mg/L, 14.3%). For the chronic kidney disease pool (ERM-DA471/IFCC-traceable target of 2.37 mg/L), the all-method mean (SD, %CV) was 2.258 mg/L (0.288 mg/L, 12.8%). There were substantial method-specific biases (mean milligram per liter reported for the normal pool was 0.780 for Siemens, 0.870 for Gentian, 0.967 for Roche, 1.061 for Diazyme, and 0.970 for other/not specified reagents; and mean milligram per liter reported for the chronic kidney disease pool was 2.052 for Siemens, 2.312 for Gentian, 2.247 for Roche, 2.909 for Diazyme, and 2.413 for other/not specified reagents). CONCLUSIONS: Manufacturers need to improve the accuracy of cystatin C measurement procedures if cystatin C is to achieve its full potential as a biomarker for estimating glomerular filtration rate.

The effects of freeze-thaw on ß-trace protein and ß2-microglobulin assays after long-term sample storage.

OBJECTIVES: To evaluate the effect of a freeze-thaw cycle on ß-trace protein (ßTP) and ß2-microglobulin (ß2M). DESIGN AND METHODS: We compared ßTP and ß2M concentrations before and after a single freeze-thaw cycle in long-term stored samples from 172 participants of the Third National Health and Nutrition Examination Survey (NHANES III). RESULTS: Measurements of ßTP and ß2M before and after freeze-thaw were highly correlated with Spearman's coefficients of 0.90 and 0.99, respectively. Serum concentrations of ßTP were slightly lower after freeze-thaw (-0.05 mg/L, P=0.006). Measurements of ß2M did not differ before and after freeze-thaw (P=0.35). CONCLUSIONS: ßTP and ß2M measurements were robust to a single freeze-thaw cycle, although ß2M appeared more stable than ßTP. These results have implications for future studies of these biomarkers.

Determining stability of stored samples retrospectively: the validation of glycated albumin.

BACKGROUND: Determining the stability of stored samples for assays that were not available at the time of original collection is problematic. To assess sample stability for a relatively new assay of glycated albumin (GA), we first measured GA in fresh samples and in samples stored for 19-23 years. We then compared the regression of the contemporaneous glycohemoglobin (Hb A(1c)) values against the GA results from fresh vs stored samples, reasoning that similar slopes and intercepts would provide strong, albeit indirect, support for the stability of the stored samples for GA measurements. METHODS: We assayed 90 samples frozen for 19-23 years and 90 fresh samples from participants in the Diabetes Control and Complications trial cohort for GA. Hb A(1c) was measured contemporaneously in fresh samples at each time period. A single normal-errors linear model regressed the Hb A(1c) values on the GA, with an additional effect for collection period (fresh vs stored for GA) and the interaction of period and GA. RESULTS: Analysis of the regressions lines between GA and Hb A(1c) revealed intercepts (3.69 and 2.97 for the fresh and stored samples, respectively) and slopes (0.198 vs 0.187) that were not significantly different (P = 0.182 and P = 0.639, respectively). CONCLUSIONS: This simple approach can be used to assess the stability of stored samples in new assays. Samples stored for as long as 23 years are suitable for the GA assay.