Johanna E. Camara

Development of a Standard Reference Material for Metabolomics Research

The National Institute of Standards and Technology (NIST), in collaboration with the National Institutes of Health (NIH), has developed a Standard Reference Material (SRM) to support technology development in metabolomics research. SRM 1950 Metabolites in Human Plasma is intended to have metabolite concentrations that are representative of those found in adult human plasma. The plasma used in the preparation of SRM 1950 was collected from both male and female donors, and donor ethnicity targets were selected based upon the ethnic makeup of the U.S. population. Metabolomics research is diverse in terms of both instrumentation and scientific goals. This SRM was designed to apply broadly to the field, not toward specific applications. Therefore, concentrations of approximately 100 analytes, including amino acids, fatty acids, trace elements, vitamins, hormones, selenoproteins, clinical markers, and perfluorinated compounds (PFCs), were determined. Value assignment measurements were performed by NIST and the Centers for Disease Control and Prevention (CDC). SRM 1950 is the first reference material developed specifically for metabolomics research.

Expression and characterization of 15N-labeled human C-reactive protein in Escherichia coli and Pichia pastoris for use in isotope-dilution mass spectrometry

Levels of C-reactive protein (CRP) in serum are correlated with inflammation and disease in humans. A higher level quantitative method, such as isotope-dilution mass spectrometry (ID-MS) is needed to compare and standardize the many commercial CRP assays. We compare the expression and purification of (15)N-CRP from Escherichia coli and Pichia pastoris and show that the protein isolated from P. pastoris has native pentameric structure along with high isotopic enrichment as shown by software developed specifically for this purpose. When this preparation was mixed in various ratios with unlabeled CRP and tryptic peptides of the mixtures were analyzed by LC-MS/MS, the ratios of heavy and light peaks were tightly correlated with input amounts of each protein. In this report we confirm the suitability of (15)N-rCRP as an internal standard in ID-MS. Standardization of CRP assays should help validate the relationship between CRP and human health.

IFCC Working Group Recommendations for Assessing Commutability Part 1: General Experimental Design

Commutability is a property of a reference material (RM) that relates to the closeness of agreement between results for an RM and results for clinical samples (CSs) when measured by ≥2 measurement procedures (MPs). Commutability of RMs used in a calibration traceability scheme is an essential property for them to be fit for purpose. Similarly, commutability of trueness controls or external quality assessment samples is essential when those materials are used to assess trueness of results for CSs. This report is part 1 of a 3-part series describing how to assess commutability of RMs. Part 1 defines commutability and addresses critical components of the experimental design for commutability assessment, including selection of individual CSs, use of pooled CSs, qualification of MPs for inclusion, establishing criteria for the determination that an RM is commutable, generalization of commutability conclusions to future measurements made with the MPs included in the assessment, and information regarding commutability to be included in the certificate for an RM. Parts 2 and 3 in the series present 2 different statistical approaches to commutability assessment that use fixed criteria related to the medical decisions that will be made using the laboratory test results.

IFCC Working Group Recommendations for Assessing Commutability Part 2: Using the Difference in Bias Between a Reference Material and Clinical Samples

A process is described to assess the commutability of a reference material (RM) intended for use as a calibrator, trueness control, or external quality assessment sample based on the difference in bias between an RM and clinical samples (CSs) measured using 2 different measurement procedures (MPs). This difference in bias is compared with a criterion based on a medically relevant difference between an RM and CS results to make a conclusion regarding commutability. When more than 2 MPs are included, the commutability is assessed pairwise for all combinations of 2 MPs. This approach allows the same criterion to be used for all combinations of MPs included in the assessment. The assessment is based on an error model that allows estimation of various random and systematic sources of error, including those from sample-specific effects of interfering substances. An advantage of this approach is that the difference in bias between an RM and the average bias of CSs at the concentration (i.e., amount of substance present or quantity value) of the RM is determined and its uncertainty estimated. An RM is considered fit for purpose for those MPs for which commutability is demonstrated.

Determination of fortified and endogenous folates in food-based Standard Reference Materials by liquid chromatography-tandem mass spectrometry

The National Institute of Standards and Technology (NIST) is developing a wide variety of Standard Reference Materials (SRMs) to support measurements of vitamins and other nutrients in foods. Previously, NIST has provided SRMs with values assigned for the folate vitamer, folic acid (pteroylglutamic acid), which is fortified in several foods due to its role in prevention of neural tube defects. In order to expand the number of food-based SRMs with values assigned for folic acid, as well as additional endogenous folates, NIST has developed methods that include trienzyme digestion and isotope-dilution liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis. Sample preparation was optimized for each individual food type, but all samples were analyzed under the same LC-MS/MS conditions. The application of these methods resulted in folic acid values for SRM 1849a Infant/Adult Nutritional Formula and SRM 3233 Fortified Breakfast Cereal of (2.33 ± 0.06) μg/g and (16.0 ± 0.7) μg/g, respectively. In addition, the endogenous folate vitamer 5-methlytetrahydrofolate (5-MTHF) was detected and quantified in SRM 1849a Infant/Adult Nutritional Formula, candidate SRM 1549a Whole Milk Powder, and candidate SRM 1845a Whole Egg Powder, resulting in values of (0.0839 ± 0.0071) μg/g, (0.211 ± 0.014) μg/g, and (0.838 ± 0.044) μg/g, respectively. SRM 1849a Infant/Adult Nutritional Formula is the first food-based NIST SRM to possess a reference value for 5-MTHF and the first certified reference material to have an assigned 5-MTHF value based on LC-MS/MS. The values obtained for folic acid and 5-MTHF by LC-MS/MS will be incorporated into the final value assignments for all these food-based SRMs.

Baseline Assessment of 25-Hydroxyvitamin D Assay Performance: A Vitamin D Standardization Program (VDSP) Interlaboratory Comparison Study.

The Vitamin D Standardization Program (VDSP) coordinated an interlaboratory study to assess the comparability of measurements of total 25-hydroxyvitamin D [25(OH)D] in human serum, which is the primary marker of vitamin D status. A set of 50 individual donor samples were analyzed by 15 different laboratories representing national nutrition surveys, assay manufacturers, and clinical and/or research laboratories to provide results for total 25(OH)D using both immunoassays (IAs) and LC tandem MS (MS/MS). The results were evaluated relative to bias compared with the target values assigned based on a combination of measurements at Ghent University (Belgium) and the U.S. National Institute of Standards and Technology using reference measurement procedures for the determination of 25(OH)D2 and 25(OH)D3. CV and mean bias for each laboratory and assay platform were assessed and compared with previously established VDSP performance criteria, namely CV ≤ 10% and mean bias ≤ 5%. Nearly all LC-MS/MS results achieved VDSP criteria, whereas only 50% of IAs met the criterion for a ≤10% CV and only three of eight IAs achieved the ≤5% bias. These results establish a benchmark for the evaluation of 25(OH)D assay performance and standardization activities in the future.

IFCC Working Group Recommendations for Assessing Commutability Part 3: Using the Calibration Effectiveness of a Reference Material

A process is described to assess the commutability of a reference material (RM) intended for use as a calibrator based on its ability to fulfill its intended use in a calibration traceability scheme to produce equivalent clinical sample (CS) results among different measurement procedures (MPs) for the same measurand. Three sources of systematic error are elucidated in the context of creating the calibration model for translating MP signals to measurand amounts: calibration fit, calibrator level trueness, and commutability. An example set of 40 CS results from 7 MPs is used to illustrate estimation of bias and variability for each MP. The candidate RM is then used to recalibrate each MP, and its effectiveness in reducing the systematic error among the MPs within an acceptable level of equivalence based on medical requirements confirms its commutability for those MPs. The RM is declared noncommutable for MPs for which, after recalibration, the CS results do not agree with those from other MPs. When a lack of agreement is found, other potential causes, including lack of calibration fit, should be investigated before concluding the RM is noncommutable. The RM is considered fit for purpose for those MPs where commutability is demonstrated.

General Steps to Standardize the Laboratory Measurement of Serum Total 25-Hydroxyvitamin D.

The Vitamin D Standardization Program (VDSP) has collaborated with numerous groups and agencies to assemble a set of tools, i.e., a reference measurement system, that can be used to establish the traceability of 25-hydroxyvitamin D [25(OH)D] assays to relevant reference measurement procedures and reference materials. This is done with the goal of verifying end-user laboratory performance using precise statistical criteria to determine whether a specific assay is standardized. The purpose of this paper was to outline a set of steps that routine clinical and research laboratories can use to standardize their 25(OH)D assays using these tools. These steps apply to laboratories using commercially developed immunoassay measurement systems as well as in-house assays, usually based on high HPLC or LC tandem MS measurement systems. The steps are (1) initial calibration, (2) initial assessment of accuracy and bias, (3) assessment of total percent CV and mean bias, (4) use of trueness controls, and (5) participation in accuracy-based performance testing and/or external quality assessment schemes. The goal of each laboratory assay is to have a total CV of ≤10% and mean bias of ≤5%. Rigorous and less rigorous but low-cost options for meeting these statistical criteria are provided. Research laboratories who infrequently measure 25(OH)D are advised to repeat steps 1-4 for every measurement cycle. For users of commercial immunoassays who have relatively little control over standardization, we present an option for using trueness controls to develop a master equation that can be used to standardize results to the reference methods.

Development of an Improved Standard Reference Material for Vitamin D Metabolites in Human Serum

The National Institute of Standards and Technology (NIST) has developed Standard Reference Material (SRM) 972a Vitamin D Metabolites in Frozen Human Serum as a replacement for SRM 972, which is no longer available. SRM 972a was developed in collaboration with the National Institutes of Healths Office of Dietary Supplements. In contrast to the previous reference material, three of the four levels of SRM 972a are composed of unmodified human serum. This SRM has certified and reference values for the following 25-hydroxyvitamin D [25(OH)D] species: 25(OH)D2, 25(OH)D3, and 3-epi-25(OH)D3. The value assignment and certification process included three isotope-dilution mass spectrometry approaches, with measurements performed at NIST and at the Centers for Disease Control and Prevention (CDC). The value assignment methods employed have been modified from those utilized for the previous SRM, and all three approaches now incorporate chromatographic resolution of the stereoisomers, 25(OH)D3 and 3-epi-25(OH)D3.

Baseline Assessment of 25-Hydroxyvitamin D Reference Material and Proficiency Testing/External Quality Assurance Material Commutability: A Vitamin D Standardization Program Study

The Vitamin D Standardization Program (VDSP) coordinated a study in 2012 to assess the commutability of reference materials and proficiency testing/external quality assurance materials for total 25-hydroxyvitamin D [25(OH)D] in human serum, the primary indicator of vitamin D status. A set of 50 single-donor serum samples as well as 17 reference and proficiency testing/external quality assessment materials were analyzed by participating laboratories that used either immunoassay or LC-MS methods for total 25(OH)D. The commutability test materials included National Institute of Standards and Technology Standard Reference Material 972a Vitamin D Metabolites in Human Serum as well as materials from the College of American Pathologists and the Vitamin D External Quality Assessment Scheme. Study protocols and data analysis procedures were in accordance with Clinical and Laboratory Standards Institute guidelines. The majority of the test materials were found to be commutable with the methods used in this commutability study. These results provide guidance for laboratories needing to choose appropriate reference materials and select proficiency or external quality assessment programs and will serve as a foundation for additional VDSP studies.