Extent of Equivalence of Results for Urine Albumin among 3 Candidate Mass Spectrometry Reference Measurement Procedures.

BACKGROUND: Urine albumin (UA) is an important biomarker of chronic kidney disease. Current in vitro diagnostic medical devices (IVD-MDs) for measuring UA are not standardized, and median results among IVD-MDs differ by approximately 45%. Since fixed decision values are used to interpret UA, higher-order reference measurement procedures (RMPs) are needed for metrological traceability. Three candidate liquid chromatography-tandem mass spectrometry RMPs have been developed for UA. METHODS: Eight single-donation human urine samples were measured by 3 candidate RMPs. Results were compared using t-test and variance component analysis. RESULTS: The mean results for each urine sample from each RMP laboratory were not statistically different from the overall mean value by t-test. The median total CV including contributions from bias and imprecision among the 3 RMP laboratories was 6.23% using variance component analysis for each sample. The allowable bias to the RMP for an end-user IVD-MD was ≦9.0% or ≦3.0% based on the desirable or optimal total allowable error of 30% or 24%, respectively. A maximum allowable standard uncertainty for an RMP result was determined to be 4.3% or 3.3% for desirable or optimal performance, respectively. The standard uncertainties for all of the RMP laboratories meet the desirable and optimal standard uncertainty specifications. CONCLUSION: The candidate RMPs for UA in these 3 laboratories have suitable agreement of results and uncertainties for use as higher-order RMPs in the metrological traceability of end-user IVD-MDs for measuring UA.

Expedient measurement of total protein in human serum and plasma via the biuret method using fiber optic probe for patient samples and certified reference materials.

The biuret method is currently recognized as a reference measurement procedure for serum/plasma total protein by the Joint Committee for Traceability in Laboratory Medicine (JCTLM). However, as the reaction involved in this method is highly time-dependent, to ensure identical measurement conditions for calibrator and samples for high accuracy, a fast and simple measurement procedure is critical to ensure the precision and trueness of this method. We measured serum/plasma total protein using a Cary 60 spectrophotometer coupled with a fiber optic probe, which was faster and simpler than the conventional cuvette method. The biuret method utilizing alkaline solutions of copper sulfate and potassium sodium tartrate was added to the sample and calibrator (NIST SRM 927e) incubated for 1 h before measurement. A panel of samples consisting of pooled human serum, single donor serum, and certified reference materials (CRMs) from three sources were measured for method validation. Sixteen native patient samples were measured using the newly developed biuret method and compared against clinical analyzers. Additionally, the results of three cycles of a local External Quality Assessment (EQA) Programme submitted by participating clinical laboratories were compared against the biuret method. Our biuret method using fiber optic probe demonstrated good precision with within-day relative standard deviation (RSD) of 0.04 to 0.23% and between-day RSD of 0.58%. The deviations between the obtained values and the certified values for all three CRMs ranged from -0.38 to 1.60%, indicating good method trueness. The routine methods using clinical analyzers were also found to agree well with the developed biuret method using fiber optic probe for EQA samples and native patient samples. The biuret method using a fiber optic probe represented a convenient and reliable way of measuring serum total protein. It also demonstrated excellent precision and trueness using CRMs and patient samples, which made the method a simpler candidate reference method for serum protein measurement.

Evaluation of low-density lipoprotein cholesterol equations by cross-platform assessment of accuracy-based EQA data against SI-traceable reference value.

OBJECTIVES: Low-density lipoprotein cholesterol (LDLC) is the primary cholesterol target for the diagnosis and treatment of cardiovascular disease (CVD). Although beta-quantitation (BQ) is the gold standard to determine LDLC levels accurately, many clinical laboratories apply the Friedewald equation to calculate LDLC. As LDLC is an important risk factor for CVD, we evaluated the accuracy of Friedewald and alternative equations (Martin/Hopkins and Sampson) for LDLC. METHODS: We calculated LDLC based on three equations (Friedewald, Martin/Hopkins and Sampson) using the total cholesterol (TC), triglycerides (TG), and high-density lipoprotein cholesterol (HDLC) in commutable serum samples measured by clinical laboratories participating in the Health Sciences Authority (HSA) external quality assessment (EQA) programme over a 5 years period (number of datasets, n=345). LDLC calculated from the equations were comparatively evaluated against the reference values, determined from BQ-isotope dilution mass spectrometry (IDMS) with traceability to the International System of Units (SI). RESULTS: Among the three equations, Martin/Hopkins equation derived LDLC had the best linearity against direct measured (y=1.141x - 14.403; R2=0.8626) and traceable LDLC (y=1.1692x - 22.137; R2=0.9638). Martin/Hopkins equation (R2=0.9638) had the strongest R2 in association with traceable LDLC compared with the Friedewald (R2=0.9262) and Sampson (R2=0.9447) equation. The discordance with traceable LDLC was the lowest in Martin/Hopkins (median=-0.725%, IQR=6.914%) as compared to Friedewald (median=-4.094%, IQR=10.305%) and Sampson equation (median=-1.389%, IQR=9.972%). Martin/Hopkins was found to result in the lowest number of misclassifications, whereas Friedewald had the most numbers of misclassification. Samples with high TG, low HDLC and high LDLC had no misclassification by Martin/Hopkins equation, but Friedewald equation resulted in ∼50% misclassification in these samples. CONCLUSIONS: The Martin/Hopkins equation was found to achieve better agreement with the LDLC reference values as compared to Friedewald and Sampson equations, especially in samples with high TG and low HDLC. Martin/Hopkins derived LDLC also enabled a more accurate classification of LDLC levels.

Commutability assessment of human urine certified reference materials for albumin and creatinine on multiple clinical analyzers using different statistical models.

Urine albumin concentration and albumin-creatinine ratio are important for the screening of early-stage kidney damage. Commutable urine certified reference materials (CRMs) for albumin and creatinine are necessary for standardization of urine albumin and accurate measurement of albumin-urine ratio. Two urine CRMs for albumin and creatinine with certified values determined using higher-order reference measurement procedures were evaluated for their commutability on five brands/models of clinical analyzers where different reagent kits were used, including Roche Cobas c702, Roche Cobas c311, Siemens Atellica CH, Beckman Coulter AU5800, and Abbott Architect c16000. The commutability study was conducted by measuring at least 26 authentic patient urine samples and the human urine CRMs using both reference measurement procedures and the routine methods. Both the linear regression model suggested by the Clinical and Laboratory Standard Institute (CLSI) guidelines and log-transformed model recommended by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Commutability Working Group were used to evaluate the commutability of the human urine CRMs. The commutability of the human urine CRMs was found to be generally satisfactory on all five clinical analyzers for both albumin and creatinine, suggesting that they are suitable to be used routinely by clinical laboratories as quality control or for method validation of urine albumin and creatinine measurements.

Candidate High-Resolution Mass Spectrometry-Based Reference Method for the Quantification of Procalcitonin in Human Serum Using a Characterized Recombinant Protein as a Primary Calibrator.

Procalcitonin (PCT) is a widely used biomarker for rapid sepsis diagnosis and antibiotic stewardship. Variability of results in commercial assays has highlighted the need for standardization of PCT measurements. An antibody-free candidate reference measurement procedure (RMP) based on the isotope dilution mass spectrometry and protein calibration approach was developed and validated to quantify PCT in human serum. The method allows quantification of PCT from 0.25 to 13.74 µg/L (R > 0.998) with extension up to 132 µg/L after dilution of samples with PCT concentration above 13.74 µg/L. Intraday bias was between -3.3 and +5.7%, and interday bias was between -3.0 and -0.7%. Intraday precision was below 5.1%, and interday precision was below 4.0%. The candidate RMP was successfully applied to the absolute quantification of PCT in five frozen human serum pools. A recombinant PCT used as a primary calibrator was characterized by high-resolution mass spectrometry and amino acid analysis to establish traceability of the results to the SI units. This candidate RMP is fit to assign target values to secondary certified reference materials (CRMs) for further use in external quality assessment schemes to monitor the accuracy and comparability of the commercially available immunoassay results and to confirm the need for improving the harmonization of PCT assays. The candidate RMP will also be used to evaluate whether the correlation between the candidate RMP and immunoassays is sufficiently high. Overall, this candidate RMP will support reliable sepsis diagnosis and guide treatment decisions, patient monitoring, and outcomes.

Measurement of urine albumin by liquid chromatography-isotope dilution tandem mass spectrometry and its application to value assignment of external quality assessment samples and certification of reference materials.

OBJECTIVES: Urine albumin is measured in clinical laboratories by immunoturbidimetry. However, large biases are observed among the different routine methods. To standardize the measurement of urine albumin, a reference measurement procedure (RMP) and urine albumin certified reference materials (CRMs) are needed. METHODS: A candidate RMP for urine albumin based on liquid chromatography-isotope dilution tandem mass spectrometry (LC-IDMS/MS) using human serum albumin as calibration standard was developed. Isotope-labeled human albumin was used as internal standard. Urine samples were digested using trypsin and eight resulting "signature" peptides of albumin were quantified by LC-IDMS/MS. The candidate RMP was employed in value assignment of external quality assessment (EQA) samples and certification of urine albumin reference materials. The commutability of the developed CRMs was assessed against patient samples. RESULTS: The candidate RMP (recovery 101.5-103.2% and CV 1.2-3.3% at about 7-40 mg/L) met optimal performance goal. The lower limit of quantification was 0.03 mg/L as determined by signal-to-noise method. The EQA results from clinical laboratories using different immunoturbidimetric methods were generally comparable with assigned target values determined by the candidate RMP, with albumin concentrations ranging from 5 to 226 mg/L. Urine albumin reference materials (two levels) certified using the candidate RMP showed good commutability in a preliminary study. CONCLUSIONS: With optimal method precision and trueness, as well as comparability with routine methods, the developed RMP may be used for value assignment of EQA samples or certification of reference materials, which are important pillars in urine albumin method standardization.

Determination of purity values of amino acid reference materials by mass balance method: an approach to the quantification of related structure impurities.

A systematic procedure for the determination of purity values of amino acid reference materials was developed by use of mass balance method where four categories of impurities (related structure impurities (RSIs), water, organic solvent residue (OSR), and non-volatile residue (NVR)) were quantified separately. The amount of RSIs was determined using a combination of three quantification methods. To ensure metrological traceability in the determination of RSIs, at least one such impurity in each candidate amino acid reference material was quantified using liquid chromatography-isotope dilution tandem mass spectrometry (LC-IDMS/MS). Other RSIs were determined using external calibration liquid chromatography-tandem mass spectrometry (LC-MS/MS) or o-phthaldialdehyde (OPA) derivatization, followed by liquid chromatography-ultraviolet (LC-UV) measurement. As the UV absorption of most RSIs came basically from the same chromophore after OPA derivatization, a relative peak area approach was used in the LC-UV method to quantify the amount of RSIs by comparing their peak areas with that of a reference RSI. The reference RSI was pre-selected and the amount determined by LC-IDMS/MS separately. The absence of D-amino acids was confirmed using Marfey's reagent derivatization, followed by LC-UV analysis. The amounts of water, OSR, and NVR were measured using Karl Fischer coulometry, gas chromatography-mass spectrometry (GC-MS) and thermogravimetry, respectively. By using this procedure, four amino acid (L-valine, L-leucine, L-isoleucine, and L-phenylalanine) certified reference materials (CRMs) were developed from the candidate materials. The homogeneity and stability of the CRMs were demonstrated by use of LC-IDMS/MS or OPA-LC-UV method, following the principles in ISO 17034 and ISO Guide 35.Graphical abstract.

Commutable whole blood reference materials for hemoglobin A1c validated on multiple clinical analyzers.

Background The measurement of hemoglobin A1c (HbA1c) is important for diagnosing diabetes mellitus as well as assessing glycemic control in diabetic patients. Commutable whole blood certified reference materials (CRMs) are needed in the measurement of HbA1c for method validation and/or as quality controls. Methods We developed three levels of hemolyzed whole blood CRMs for HbA1c. The certified values were determined using liquid chromatography-isotope dilution tandem mass spectrometry method (LC-IDMS/MS) where two "signature" hexapeptides of HbA1c and hemoglobin A0 (HbA0) were used as the calibration standards. The concentrations of the hexapeptide solutions were determined by amino acid analysis by the LC-IDMS/MS method using amino acid CRMs as the calibration standards. The commutability study was conducted by measuring 25 patient specimens and the whole blood CRMs by both LC-IDMS/MS method and various routine methods using six different clinical analyzers. Results The certified values were determined to be 35.1±2.0, 50.3±1.9 and 65.8±2.6 mmol/mol, respectively. These CRMs showed good commutability on five of the six clinical analyzers but showed poor commutability on one of the clinical analyzers that used similar method as two other analyzers where good commutability was observed. Conclusions With certified target values based on metrological traceability and good commutability on most of the clinical analyzers, the developed whole blood CRMs can be used for method validation or as quality control materials in the measurement of HbA1c. The commutability study results also underscored the need of commutability testing of clinical CRMs using various clinical analyzers.

Simple and accurate candidate reference measurement procedure for total testosterone in human serum by one-step liquid-liquid extraction coupled with isotope dilution mass spectrometry.

Testosterone in human serum is commonly tested in clinical laboratories using immunoassay methods as well as liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. To standardize and ensure the accuracy of the measurement results, reference procedures with higher metrological order are required. A simple measurement procedure based on one-step liquid-liquid extraction (LLE) and liquid chromatography-isotope dilution tandem mass spectrometry (LC-IDMS/MS) was developed for total testosterone in human serum. The procedure involved serum spiked with 13C3-testosterone, equilibration for 2 h, and extraction with an organic solvent. Testosterone certified reference material (CRM) was used as the calibration standard to ensure the traceability to the International System of Units (SI). Testosterone in serum CRMs from the National Institute for Standards and Technology (NIST) and LGC were used to validate the accuracy of the newly developed method. The deviations of the obtained values from the NIST and LGC certified values ranged from -0.55% to 0.45%. Similarly, the coefficient of variations (CVs) of the replicate measurements were in the range of 0.55% and 0.78%, respectively. The relative expanded uncertainties were comparable with those of the certified materials. The newly developed LC-IDMS/MS procedure demonstrated adequate trueness and precision, and was simple to perform. The method can be used for value assignment of testosterone in external quality assessment (EQA) materials as well as certification of CRMs in the future. Graphical abstract.

Enhancing the accuracy of measurement of small molecule organic biomarkers.

Over two decades, the Organic Analysis Working Group (OAWG) of the Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology (CCQM) has organized a number of comparisons for clinically relevant small molecule organic biomarkers. The aim of the OAWG community is to be part of the coordinated international movement towards accuracy and comparability of clinical measurements that will, in turn, minimize the wastage of repeat testing and unnecessary therapy to create a sustainable healthcare industry. International and regional directives/requirements on metrological traceability of calibrators and control materials are in place. Metrology institutes worldwide maintain infrastructure for the practical realization of metrological traceability and demonstrate the equivalence of their measurement capabilities through participation in key comparisons organized under the auspices of the CCQM. These institutes provide certified reference materials, as well as other dedicated value-assignment services benefiting the in-vitro diagnostic (IVD) industry, reference (calibration) laboratories and the clinical chemistry laboratories. The roles of these services in supporting national, regional, and international activities to ensure the metrological traceability of clinical chemistry measurements are described. Graphical abstract.