Ashley Beasley Green (Presenter)
NIST
Authorship: Ashley Beasley-Green, David Bunk, Karen Phinney
National Institute of Standards and Technology
| Short Abstract Urinary excretion of albumin is a major diagnostic and prognostic marker of renal dysfunction and cardiovascular disease; therefore, accurate measurement of urine albumin is vital to clinical diagnosis. To address urine albumin measurement precision, we have developed the following components of the urine albumin reference measurement system: a multiplexed candidate reference measurement procedure that utilizes isotope dilution-mass spectrometry (ID-MS) and multiple reaction monitoring (MRM) to quantify urine albumin; a primary reference material to be used as a calibrator for higher-order urine albumin methods; and a secondary reference material to be used as a matrix-based quality control for commercially-available urine albumin assays. |
Long Abstract
Urine albumin and urine albumin/creatinine ratios are used in the detection, treatment, and monitoring of chronic kidney disease. Although inter-method differences and analyte heterogeneity have been reported for urine albumin measurements, accuracy assessments of the available methods have been hindered by the lack of a reference measurement system, including reference measurement procedures and reference materials, for this clinical analyte. The development of a urine albumin reference measurement system will create a chain of traceability that will link routine clinical measurements to SI units. Therefore, we have developed the following components of the urine albumin reference measurement system: a multiplexed candidate reference measurement procedure that utilizes isotope dilution-mass spectrometry (ID-MS) and multiple reaction monitoring (MRM) to quantify urine albumin; a primary reference material to be used as a calibrator for higher-order urine albumin methods; and a secondary reference material to be used as a matrix-based quality control for commercially-available urine albumin assays.
The candidate reference measurement procedure is a MS-based multiplexed assay that incorporates an isotopically-labeled (15N) full-length recombinant human serum albumin material as the internal standard, which permits the absolute quantitation of full-length albumin in human urine. A total of 11 peptides with two transitions per peptide (22 MRM transitions) were selected from the tryptic digestion of human serum albumin on the basis of retention time reproducibility, peak intensity, and the degree of HSA sequence coverage. Measurement reproducibility and repeatability and the within/between peptide comparability were evaluated to assess the validity of the urine albumin measurements. The primary reference material, a highly characterized recombinant human serum albumin (HSA) standard reference material (SRM 2925), was incorporated into the multiplexed assay to evaluate measurement reproducibility and repeatability and the effect of the MRM transition/peptide measurements on the total urine albumin content. Pooled and non-pooled patient urine samples were analyzed to assess the influence of biological variability on the precision of the multiplexed urine albumin assay.
MS-based quantification of urine albumin provides both accurate and repeatable measurements at both micro- and normoalbuminuria levels, which can facilitate early diagnosis of kidney dysfunction. In addition to the quantitative advantages, we are also able to qualitatively evaluate molecular heterogeneity of endogenous urine albumin via the incorporation of multiple peptides that span the HSA sequence in the multiplexed assay. The high degree of selectivity and sensitivity of the MS-based urine albumin assay coupled with the highly purified SRM 2925 calibrator will support the value-assignment efforts of the matrix-based urine albumin secondary reference material (SRM 3666), which can be used as a quality assessment tool for clinical laboratories or assay manufacturers to verify the accuracy of their urine albumin measurements.
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