Higher Order Reference Measurement Procedures Research Articles

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

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. Eight single-donation human urine samples were measured by 3 candidate RMPs. Results were compared using t-test and variance component analysis. 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. 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.

On Standardization and Evaluation of Continuous Glucose Monitoring Systems

Continuous glucose monitoring (CGM) systems are often used to monitor blood glucose levels. Most commercially available CGM systems continuously measure glucose concentrations in the interstitial fluid of subcutaneous adipose tissue. However, there is currently no internationally accepted reference method for measuring interstitial fluid glucose, which is a prerequisite for metrological traceability of glucose measurements obtained using CGM. Since manufacturers do not provide information about the traceability chain and measurement uncertainty of their systems, CGM-derived glucose values cannot currently be adequately traced to standards or higher order reference measurement procedures. Additionally, the «mean absolute relative difference» (MARD) often used to describe the analytical performance of CGM systems is dependent on many factors. For example, the MARD can be significantly affected by the «lag time» between the change in blood glucose and interstitial glucose, especially at high rates of change in glucose. Finally, modern automated insulin delivery (ADI) systems with integrated CGM can automatically suspend or increase insulin infusion in response to current and/or predicted hypoglycemic and hyperglycemic phenomenon in children and adults with type 1 diabetes mellitus (T1DM). The purpose of the review is justification of the necessity to establish metrological traceability of glucose measurements with CGM systems, as well as a discussion of the analytical and clinical characteristics of CGM systems proposed by various professional communities. Based on the results of the review, it was concluded that it is necessary to 1) develop metrological support for glucose measurements performed using CGM systems, 2) solve the problems of ensuring the accessibility and usability of CGM systems by patients in real conditions.

Development of an LC-MRM-MS-Based Candidate Reference Measurement Procedure for Standardization of Serum Apolipoprotein (a) Tests.

Medical results generated by European CE Marking for In Vitro Diagnostic or in-house tests should be traceable to higher order reference measurement systems (RMS), such as International Federation of Clinical Chemistry and Laboratory Medicine (IFCC)-endorsed reference measurement procedures (RMPs) and reference materials. Currently, serum apolipoprotein (a) [apo(a)] is recognized as a novel risk factor for cardiovascular risk assessment and patient management. The former RMS for serum apo(a) is no longer available; consequently, an International System of Units (SI)-traceable, ideally multiplexed, and sustainable RMS for apo(a) is needed. A mass spectrometry (MS)-based candidate RMP (cRMP) for apo(a) was developed using quantitative bottom-up proteomics targeting 3 proteotypic peptides. The method was provisionally validated according to ISO 15193 using a single human serum based calibrator traceable to the former WHO-IFCC RMS. The quantitation of serum apo(a) was by design independent of its size polymorphism, was linear from 3.8 to 456 nmol/L, and had a lower limit of quantitation for apo(a) of 3.8 nmol/L using peptide LFLEPTQADIALLK. Interpeptide agreement showed Pearson Rs of 0.987 and 0.984 for peptides GISSTVTGR and TPENYPNAGLTR, and method comparison indicated good correspondence (slopes 0.977, 1.033, and 1.085 for LFLEPTQADIALLK, GISSTVTGR, and TPENYPNAGLTR). Average within-laboratory imprecision of the cRMP was 8.9%, 11.9%, and 12.8% for the 3 peptides. A robust, antibody-independent, MS-based cRMP was developed as higher order RMP and an essential part of the apo(a) traceability chain and future RMS. The cRMP fulfils predefined analytical performance specifications, making it a promising RMP candidate in an SI-traceable MS-based RMS for apo(a).

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. Boththe 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.

Evaluation of the necessity and the feasibility of the standardization of procalcitonin measurements: Activities of IFCC WG-PCT with involvement of all stakeholders

Procalcitonin (PCT) is an important biomarker for sepsis diagnosis and management. To date, there is no higher-order reference measurement procedure (RMP) and certified reference material to achieve global standardization of results and results traceability to the SI units. Although efforts have been made to harmonize PCT results, a number of comparison studies and external quality assessment (EQA) schemes show conflicting results regarding results comparability and to date, equivalence of PCT results across the assays remains questionable in absence of studies relying on commutable EQA materials. In this context, the IFCC initiated activities to fill these gaps through the creation of the working group on standardization of PCT assays that gathers experts from National Metrology Institutes, calibration laboratories, clinicians, biologists, EQA providers and assay manufacturers. Among the activities, a higher order RMP and commutable reference materials are under development to build a robust reference measurement system (RMS). A commutability study is being organized to identify EQA materials that are fit for purpose to reliably estimate the current comparability of PCT results. This work will make it possible to evaluate the necessity and the feasibility for establishing and maintaining a new RMS for PCT assays, if deemed necessary.

MON-130 Improving the Accuracy and Reliability of Free Thyroxine (FT4) Measurements Through the CDC Clinical Standardization Programs (CSP)

Reliable free thyroxine (FT4) measurements are essential for assessing thyroid function and for correctly diagnosing and treating thyroid disorders. Thyroid hormones play an important role in normal brain development of the fetus, and abnormal FT4 during pregnancy is associated with adverse pregnancy outcomes. Standardization of FT4 measurements, is critical to improving the accuracy and reliability of current methods and thus to improve diagnosis, treatment and prevention of thyroidal illnesses. Currently, there are no serum-based reference materials available for FT4 to assess the accuracy and reliability of FT4 assays. CDC CSP is collaborating with the International Federation of Clinical Chemistry and Laboratory Medicine, and the Partnership for the Accurate Testing for Hormones to address these issues through development of an accurate and sensitive higher-order Reference Measurement Procedure (RMP) for FT4 that will be used to assign target value to serum-based materials. The CDC CSP FT4 reference method is using equilibrium dialysis in combination with liquid chromatography tandem mass spectrometry (LC-MS/MS). FT4 in serum is isolated from the binding proteins in 1 mL equilibrium dialysis cells for 4 hours at 37oC. FT4 is further isolated by extractions prior to LC-MS/MS analysis. To determine the concentration of FT4 in serum, certified primary reference materials are used to prepare calibration materials. Chromatographic separation is achieved using a C18 reverse phase column with a gradient of methanol and water with 0.1% formic acid. Quantification by selective reaction monitoring is performed in the positive mode using electrospray ionization. Two transitions are monitored for each analyte and internal standard, and triplicate injections are used to minimize instrument instability. The within- and between-day imprecision for the CDC RMP are 2.2–3.9% and 1.8–2.6%, respectively. By comparisons with the RMPs at Ghent University in Belgium, Radboud University Medical Center in the Netherlands, and the Reference Material Institute for Clinical Chemistry Standards in Japan, the CDC RMP reported a bias within +2.5% of the mean for all labs. Factors affecting measurement accuracy were investigated, to maximize recovery for optimum performance of the method. FT4 was detectable in all samples, and thus, suitable for analysis of hypo-, eu-, and hyperthyroid patients. A comparison among hypo-, eu-, and hyperthyroid patients of a commercially available immunoassay and the CDC RMP indicated a mean bias of -37.7%. The CDC CSP has also evaluated the RMP for accurate measurement of FT4 in pregnant individuals. This candidate reference method for FT4 in serum demonstrates good accuracy and precision, and can be used as a viable base for accuracy to which routine methods for FT4 can be compared. 1. Van Houcke, S.K., et. al. Clin. Chem. Lab. Med. 2011, 49, 1275–1281.

MON-602 Improving The Accuracy And Reliability Of Free Thyroxine Measurements Through The CDC Clinical Standardization Programs

Reliable free thyroxine (FT4) measurements are essential for assessing thyroid function and properly diagnosing and treating thyroid disorders. Although FT4 measurements are used extensively in research and clinical practice, the accuracy and reliability of current methods prevent correct detection, treatment, and prevention of thyroid disorders in patient care, making standardization of FT4 measurements a priority. Currently, there are no serum-based reference materials commercially available for FT4. CDC Clinical Standardization Programs (CDC CSP) is working with the Committee for Standardization of Thyroid Function Tests (C-STFT) of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) and the Partnership for the Accurate Testing for Hormones (PATH) to address these issues through development of an accurate and sensitive higher-order Reference Measurement Procedure (RMP) for FT4. The CDC Clinical Reference Laboratory has developed a FT4 method using a modified equilibrium dialysis procedure. 1 FT4 in serum was isolated from the binding proteins in 1 mL equilibrium dialysis cells for 4 hours at 37oC. Dialysates were spiked with internal standard (thyroxine-13C6) and further isolated with extractions prior to injection on a liquid chromatography tandem mass spectrometry (LC-MS/MS) instrument. Bracketed calibration and primary reference materials were used to determine the concentration of FT4 in serum. Chromatographic separation was achieved using a C18 reverse phase column with a gradient of water and acetonitrile with addition of 0.1% formic acid. Quantification by selective reaction monitoring (SRM) analysis was performed in the positive ion mode using electrospray ionization (ESI). Two transitions were monitored for each analyte and internal standard, and triplicate injections were used to minimize any instrument instability. The proposed RMP has been evaluated and optimized for precision, accuracy, and sensitivity. The within-day and between-day imprecision are 2.5-2.9% and 0.9-1.3%, respectively. By comparisons with the Reference Laboratory at the University of Ghent, the proposed CDC RMP reported a bias within +1.0%. Factors affecting measurement accuracy were investigated and steps were taken to maximize recovery for optimum performance of the method. FT4 was detectable in all samples and thus suitable for analysis of hypo-, eu-, and hyperthyroid patients. To ensure accurate values are reported for FT4, careful consideration is needed for all steps of sample preparation and analysis. This candidate reference method for FT4 in serum demonstrates good accuracy and precision, and as such this method can be used as a viable base for accuracy to which routine methods for FT4 can be compared. 1. Van Houcke, S.K., et. al. Clin. Chem. Lab. Med. 2011, 49, 1275-1281.

Reference measurement procedure for the determination of electrolytes in human blood via ICP-OES measurement

The determination of electrolytes in human body fluids is one of the most frequently performed analyses in clinical routine laboratories. Metrological traceability of measurement results in patient samples is essential and requires the involvement of higher order reference measurement procedures wherever available. Here, the authors present the evaluation of a higher order reference system for the simultaneous determination of K+, Li+, Na+, Ca2+ and Mg2+ in blood serum and plasma. In the same order, the determined measurement performances were as follows: measurement ranges: 0.75 mmol l−1–75.0 mmol l−1, 0.05 mmol l−1–5.00 mmol l−1, 5 mmol l−1–200 mmol l−1, 0.4 mmol l−1–8.0 mmol l−1 and 0.1 mmol l−1–4.0 mmol l−1. Measurement imprecision: CVs were ⩽1.1% for intra assay investigations and ⩽1.8% for long term inter assay investigations for all measurands. Excellent accuracy was found testing certified Standard Reference Materials from NIST: SRM 909 (deviations from 0.0% to 1.1%) and SRM 956 (deviations from 0.0% to 1.5%). Intercomparisons with the German Metrology Institute (PTB) revealed differences from 0.1% to 0.8%. Matrix influences and carry over were not detectable. The expanded combined measurement uncertainties for the determination of the reference method values were estimated as ⩾1.5% (k = 2) for each measurand. The reference measurement procedure is accredited by the German accreditation body (DAkkS) in association with the German calibration service (DKD) according to ISO 17025 and ISO 15195. Services comprise the certification of calibrators, control materials and samples used in proficiency testing schemes.

Development of Standard Reference Materials to support assessment of iodine status for nutritional and public health purposes

The use of urinary iodine as an indicator of iodine status relies in part on the accuracy of the analytical measurement of iodine in urine. Likewise, the use of dietary iodine intake as an indicator of iodine status relies in part on the accuracy of the analytical measurement of iodine in dietary sources, including foods and dietary supplements. Similarly, the use of specific serum biomarkers of thyroid function to screen for both iodine deficiency and iodine excess relies in part on the accuracy of the analytical measurement of those biomarkers. The National Institute of Standards and Technology has been working with the NIH Office of Dietary Supplements for several years to develop higher-order reference measurement procedures and Standard Reference Materials to support the validation of new routine analytical methods for iodine in foods and dietary supplements, for urinary iodine, and for several serum biomarkers of thyroid function including thyroid-stimulating hormone, thyroglobulin, total and free thyroxine, and total and free triiodothyronine. These materials and methods have the potential to improve the assessment of iodine status and thyroid function in observational studies and clinical trials, thereby promoting public health efforts related to iodine nutrition.

Quantification of cells with specific phenotypes I: determination of CD4+ cell count per microliter in reconstituted lyophilized human PBMC prelabeled with anti-CD4 FITC antibody.

A surface-labeled lyophilized lymphocyte (sLL) preparation has been developed using human peripheral blood mononuclear cells prelabeled with a fluorescein isothiocyanate conjugated anti-CD4 monoclonal antibody. The sLL preparation is intended to be used as a reference material for CD4+ cell counting including the development of higher order reference measurement procedures and has been evaluated in the pilot study CCQM-P102. This study was conducted across 16 laboratories from eight countries to assess the ability of participants to quantify the CD4+ cell count of this reference material and to document cross-laboratory variability plus associated measurement uncertainties. Twelve different flow cytometer platforms were evaluated using a standard protocol that included calibration beads used to obtain quantitative measurements of CD4+ T cell counts. There was good overall cross-platform and counting method agreement with a grand mean of the laboratory calculated means of (301.7 ± 4.9) μL−1 CD4+ cells. Excluding outliers, greater than 90% of participant data agreed within ±15%. A major contribution to variation of sLL CD4+ cell counts was tube to tube variation of the calibration beads, amounting to an uncertainty of 3.6%. Variation due to preparative steps equated to an uncertainty of 2.6%. There was no reduction in variability when data files were centrally reanalyzed. Remaining variation was attributed to instrument specific differences. CD4+ cell counts obtained in CCQM-P102 are in excellent agreement and show the robustness of both the measurements and the data analysis and hence the suitability of sLL as a reference material for interlaboratory comparisons and external quality assessment. © 2015 The Authors. Published by Wiley Periodicals, Inc.

Validation of isotope dilution surface‐enhanced Raman scattering (IDSERS) as a higher order reference method for clinical measurands employing international comparison schemes

Isotope dilution surface‐enhanced Raman scattering (IDSERS) was established as a higher order reference measurement method for clinical measurands in international comparison schemes and in direct comparison with established mass spectrometric methods. IDSERS represents the most accurate and precise quantitative Raman technique and is capable of providing SI‐traceable amount‐of‐substance measurements. IDSERS has now been validated by participation in two international ring trials organized within the framework of the Metre Convention on the determination of biomarkers in human serum. The concentration of creatinine has been measured in two human serum samples supplied by the study organizer. The results obtained by IDSERS agree excellently with the data reported by participating national metrology institutes using mass spectrometry, and the associated uncertainties are within the claimed limits of equivalence. The performance and applicability of IDSERS were thereby convincingly demonstrated. This has led to the approval of the method as a higher order reference measurement procedure, which is now listed in a publicly accessible database maintained by the International Bureau of Weights and Measures (BIPM). Copyright © 2013 John Wiley & Sons, Ltd.

NHANES Monitoring of Serum 25-Hydroxyvitamin D: A Roundtable Summary1–3

A roundtable to discuss monitoring of serum 25-hydroxyvitamin D [25(OH)D] in the NHANES was held in late July 2009. Topics included the following: 1) options for dealing with assay fluctuations in serum 25(OH)D in the NHANES conducted between 1988 and 2006; 2) approaches for transitioning between the RIA used in the NHANES between 1988 and 2006 to the liquid chromatography tandem MS (LC-MS/MS) measurement procedure to be used in NHANES 2007 and later; 3) approaches for integrating the recently available standard reference material for vitamin D in human serum (SRM 972) from the National Institute of Standards and Technology (NIST) into the NHANES; 4) questions regarding whether the C-3 epimer of 25-hydroxyvitamin D3 [3-epi-25(OH)D3] should be measured in NHANES 2007 and later; and 5) identification of research and educational needs. The roundtable experts agreed that the NHANES data needed to be adjusted to control for assay fluctuations and offered several options for addressing this issue. The experts suggested that the LC-MS/MS measurement procedure developed by NIST could serve as a higher order reference measurement procedure. They noted the need for a commutability study for the recently released NIST SRM 972 across a range of measurement procedures. They suggested that federal agencies and professional organizations work with manufacturers to improve the quality and comparability of measurement procedures across all laboratories. The experts noted the preliminary nature of the evidence of the 3-epi-25(OH)D3 but felt that it should be measured in 2007 NHANES and later.

Standardisation of cardiac troponin I measurement: past and present

The laboratory measurement of cardiac troponin (cTn) concentration is a critical tool in the diagnosis of acute myocardial infarction (MI). Current cTnI assays produce different absolute troponin numbers and use different clinical cut-off values; hence cTnI values cannot be interchanged, with consequent confusion for clinicians. A recent Australian study compared patient results for seven cTnI assays and showed that between-method variation was approximately 2- to 5-fold. A major reason for poor method agreement is the lack of a suitable common reference material for the calibration of cTnI assays by manufacturers. Purified complexed troponin material lacks adequate commutability for all assays; hence a serum-based secondary reference material is required for cTnI with value assignment by a higher order reference measurement procedure. There is considerable debate about how best to achieve comparability of results for heterogeneous analytes such as cTnI, whether it should be via the harmonisation or the standardisation process. Whereas harmonisation depends upon consensus value assignment and uses those commercial methods which give the closest agreement at the time, standardisation comes closer to the true value through a reference measurement system that is based upon long-term calibration traceability. The current paper describes standardisation efforts by the International Federation of Clinical Chemistry and Laboratory Medicine Working Group on Standardization of cTnI (IFCC WG-TNI) to establish a reference immunoassay measurement procedure for cTnI of a higher order than current commercial immunoassay methods and a commutable secondary reference material for cTnI to which companies can reference their calibration materials.

Certification of Creatinine in a Human Serum Reference Material by GC-MS and LC-MS

To meet recommendations given by the Laboratory Working Group of the National Kidney Disease Education Program for improving serum creatinine measurements, NIST developed standard reference material (SRM) 967 Creatinine in Frozen Human Serum. SRM 967 is intended for use by laboratories and in vitro diagnostic equipment manufacturers for the calibration and evaluation of routine clinical methods. The SRM was produced from 2 serum pools with different creatinine concentrations. The concentrations were certified using a higher-order isotope-dilution GC-MS method and an isotope-dilution LC-MS method. The LC-MS method is a potential higher-order reference measurement procedure. The GC-MS mean (CV) concentrations were 67.0 (0.9%) mumol/L for serum pool 1 and 346.1 (0.45%) mumol/L for serum pool 2. The LC-MS results were 66.1 (0.2%) mumol/L and 346.3 (0.2%) mumol/L, respectively. For serum pool 1, there was a 1.4% difference between the mean GC-MS and LC-MS measurements, and a 0.10% difference for serum pool 2. The results from the 2 methods were combined to give the certified concentrations and expanded uncertainties. The certified concentration (expanded uncertainty) of SRM 967 was 66.5 (1.8) mumol/L for serum pool 1 (a value close to the diagnostically important concentration of 88.4 mumol/L) and 346.2 (7.4) mumol/L for serum pool 2 (a concentration corresponding to that expected in a patient with chronic kidney disease).

Trueness verification and traceability assessment of results from commercial systems for measurement of six enzyme activities in serum: An international study in the EC4 framework of the Calibration 2000 project

Background The in vitro diagnostics directive of the European Union requires traceability to higher order reference measurement procedures and materials for analytes in assuring the result trueness and comparability of laboratory measurements. Manufacturers must ensure that the systems they market are calibrated against available reference systems. Validation of metrologically traceable calibrations is, however, required. Methods A commutable serum-based material was analyzed in three reference laboratories and target values for six enzymes (ALT, AST, CK, GGT, LD, amylase) were assigned using IFCC reference measurement procedures. 70 laboratories in Germany, Italy, and The Netherlands measured the same enzymes in the material using procedures from six commercial companies. A system for maximum allowable error was developed from the biological variation model and the results of the various procedures were tested on their compliance to trueness and between-laboratory and within-laboratory variations relative to the maximum allowable. Results For ALT results were relatively good. > 95% of laboratories using systems from Dade, Olympus, Ortho and Roche are expected to comply traceability within the biologically derived limits, and 94% respectively 89% from Abbott and Beckman. For AST and GGT only Dade respectively Olympus fully complied. For CK all companies showed significant bias. Nevertheless > 95% of laboratories applying Abbott, Beckman and Roche systems will comply. Finally, LD and amylase measurements require significant improvement. Some manufacturers continue to sell on the European market assays giving results which are not traceable to the internationally accepted reference systems. Conclusions The traceability of enzyme measurements obtained with routine procedures to internationally accepted IFCC reference systems is not yet satisfactorily accomplished in clinical practice.