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Advances in standardization of laboratory measurement procedures: Implications for measuring biomarkers of folate and vitamin B-12 status in NHANES
Abstract
Population studies such as NHANES analyze large numbers of laboratory measurements and are often performed in different laboratories using different measurement procedures and over an extended period of time. Correct clinical and epidemiologic interpretations of the results depend on the accuracy of those measurements. Unfortunately, considerable variability has been observed among assays for folate, vitamin B-12, and related biomarkers. In the past few decades, the science of metrology has advanced considerably, with the development of improved primary reference measurement procedures and high-level reference materials, which can serve as the basis for accurate measurement. A rigorous approach has been established for making field methods traceable to the highest-level reference measurement procedures and reference materials. This article reviews some basic principles of metrology and describes their recent application to measurements of folate and vitamin B-12.
... The use of commercial QC materials with assigned values for nutritional biomarkers should not be confused with the use of certified reference materials available from metrologic agencies such as the National Institute of Standards and Technology (NIST). There are continued issues with the comparability of data across methods and laboratories (80). A major source of information for method comparability comes from PT programs. ...
... A major source of information for method comparability comes from PT programs. These programs often modify the testing materials (e.g., addition of preservatives or stabilizers, supplementation with nonnative forms of the analyte, use of animal blood or outdated human blood from blood banks), which sometimes makes the materials noncommutable for certain assays (i.e., the assay responds differently to the modified material than with native material) (80). Method comparability information derived from PT programs thus needs to be interpreted with caution. ...
For the past 45 y, the National Center for Health Statistics at the CDC has carried out nutrition surveillance of the US population by collecting anthropometric, dietary intake, and nutritional biomarker data, the latter being the focus of this publication. The earliest biomarker testing assessed iron and vitamin A status. With time, a broad spectrum of water- and fat-soluble vitamins was added and biomarkers for other types of nutrients (e.g., fatty acids) and bioactive dietary compounds (e.g., phytoestrogens) were included in NHANES. The cross-sectional survey is flexible in design, and biomarkers may be measured for a short period of time or rotated in and out of surveys depending on scientific needs. Maintaining high-quality laboratory measurements over extended periods of time such that trends in status can be reliably assessed is a major goal of the testing laboratories. Physicians, health scientists, and policy makers rely on the NHANES reference data to compare the nutritional status of population groups, to assess the impact of various interventions, and to explore associations between nutritional status and health promotion or disease prevention. Focusing on the continuous NHANES, which started in 1999, this review uses a "lessons learned" approach to present a series of challenges that are relevant to researchers measuring biomarkers in NHANES and beyond. Some of those challenges are the use of multiple related biomarkers instead of a single biomarker for a specific nutrient (e.g., folate, vitamin B-12, iron), adhering to special needs for specimen collection and handling to ensure optimum specimen quality (e.g., vitamin C, folate, homocysteine, iodine, polyunsaturated fatty acids), the retrospective use of long-term quality-control data to correct for assay shifts (e.g., vitamin D, vitamin B-12), and the proper planning for and interpretation of crossover studies to adjust for systematic method changes (e.g., folate, vitamin D, ferritin).
... Vitamin B12, the largest of all vitamins, has multiple forms and very low serum levels. It binds to serum proteins tightly [10]. All these properties of vitamin B12, difficulty of producing pure reference materials and the lack of reference method make the standardization of assays more difficult [10]. ...
... It binds to serum proteins tightly [10]. All these properties of vitamin B12, difficulty of producing pure reference materials and the lack of reference method make the standardization of assays more difficult [10]. ...
Diagnosis of vitamin B12 deficiency is generally based on the measurement of serum vitamin B12 levels. However, in selected cases functional indices of vitamin B12, such as methylmalonic acid (MMA) and homocysteine (HCY), are needed. Here we compare the performance of four automated total vitamin B12 assays and also investigate how these assays relate to functional indices of vitamin B12 status.
Total vitamin B12, MMA and HCY were measured in 69 serum samples from routine vitamin B12 assay requests. Serum vitamin B12 analysis was performed using four different immunoassay autoanalyzers: DxI 800 Unicel (Beckman Coulter, USA), ADVIA Centaur XP (Siemens Diagnostics, Tarrytown, NY, USA), Roche Cobas E601 (Roche Diagnostics, Germany), Architect i2000sr (Abbott Laboratories, Abbott Park, IL, USA). Serum MMA levels were determined by liquid chromatography-mass spectrometry (LC-MS) and serum homocysteine levels were determined by high pressure liquid chromatography (HPLC) methods.
Four immunoassay methods were comparable and correlated with each other. Correlation coefficients (r) ranged from 0.898 to 0.987, p
... Despite our attempt to make these data more comparable, our approach using proficiency testing data still has many limitations, including (1) the average percent difference between assays does not capture the sometimes large among-sample variability or a concentration-dependent bias, 21,78 (2) assay comparisons used smaller than desirable numbers of proficiency testing samples in some cases; (3) data on assay comparisons sometimes came from several years before the survey; and (4) proficiency testing samples may behave differently than native patient samples because they may have to be modified to improve their stability or allow generation of large volumes of material. 79 While in the latter case the direction of the bias is not predictable, this weakness is unlikely to have affected our interpretation because the majority of assay comparisons were based on proficiency data from the UK NEQAS Haematinics program, which uses largely unmodified materials. ...
Inadequate folate status in women of reproductive age (WRA) can lead to adverse health consequences of public health significance, such as megaloblastic anemia (folate deficiency) and an increased risk of neural tube defect (NTD)‐affected pregnancies (folate insufficiency). Our review aims to evaluate current data on folate status of WRA. We queried eight databases and the World Health Organization Micronutrients Database, identifying 45 relevant surveys conducted between 2000 and 2014 in 39 countries. Several types of folate assays were used in the analysis of blood folate, and many surveys used folate cutoffs not matched to the assay. To allow better comparisons across surveys, we attempted to account for these differences. The prevalence of folate deficiency was >20% in many countries with lower income economies but was typically <5% in countries with higher income economies. Only 11 surveys reported the prevalence of folate insufficiency, which was >40% in most countries. Overall, folate status data for WRA globally are limited and must be carefully interpreted due to methodological issues. Future surveys would benefit from using the microbiologic assay to assess folate status, along with assay‐matched cutoffs to improve monitoring and evaluation of folic acid interventions, thus informing global efforts to prevent NTDs. Inadequate folate status in women of reproductive age (WRA) can lead to adverse health consequences of public health significance, such as megaloblastic anemia (folate deficiency) and an increased risk of neural tube defect (NTD)‐affected pregnancies (folate insufficiency). Our review aims to evaluate current data on folate status of WRA. We queried eight databases and the World Health Organization Micronutrients Database, identifying 45 relevant surveys conducted between 2000 and 2014 in 39 countries.
... A pesar de los grandes avances en la automatización de los procedimientos de medición en algunos casos, y como barrera técnica, los métodos de rutina presentan una selectividad diferente a la del método de referencia, o los MR empleados por los laboratorios no son trazables al SI, lo que dificulta la comparabilidad de los resultados y la conmutabilidad entre MR (11). Figura 1. Cadena de trazabilidad (9,10). Uc (y): incertidumbre de la medición ...
The results produced by clinical laboratories are key elements in the diagnosis and treatment of diseases and to monitor patients, so, it requires a metrological control over the measurement process, which will determine the degree of comparability and trust required on the obtained measurement results. Therefore, in this paper we discuss about the comparability of measurements and how it is influenced by factors such as commutability of reference materials, measurement methods, availability of materials and reference procedures, in addition to reference intervals and decision limits for a particular measurand. Finally it is exposed what are the mechanisms that allow ensure measurements comparability on this area.
... A certain preparation becomes an RM after consensus agreement of the proficiency testing network of laboratories and it should be applicable for standardization of different methods. Production of an RM is assigned to a respectable metrology institution such as the National Institute of Standards and Technology (NIST), World Health Organization (WHO) or National Institute for Biological Standards and Control (NIBSC) [36]. The RM is distributed to final users together with a certificate declaring its characteristics (including limitations such as cross-reactivity or inapplicability for certain methods) when it becomes a certified RM (CRM). ...
Post-translational modifications (PTM) of proteins determine the activity, stability, specificity, transportability and lifespan of a protein. Some PTM are highly specific and regulated involving various enzymatic pathways, but there are other non-enzymatic PTM (nePTM), which occur stochastically, depend on the ternary structure of proteins and can be damaging. It is often observed that inactive and abnormal proteins accumulate in old cells and tissues. The nature, site and extent of nePTM give rise to a population of that specific protein with alterations in structure and function ranging from being fully active to totally inactive molecules. Determination of the type and the amount (abundance) of nePTM is essential for establishing connection between specific protein structure and specific biological role. This article summarizes analytical demands for reliable quantification of nePTM, including requirements for the assay performance, standardization and quality control, and points to the difficulties, uncertainties and un-resolved issues.
Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.
... Establishment of the appropriate limits is of particular importance when it comes to classifying patients as the population with a sub-clinical deficiency is asymptomatic. Furthermore, the lack of a gold standard complicates the diagnosis and subsequent decision-making, especially when, as in the case of a sub-clinical deficiency, diagnosis depends entirely on biomarkers [9]. The sensitivity and specificity problems associated with the vast majority of assays used to detect a vitamin B12 deficiency (cobalamine, methylmalonic acid, holotranscobalamine II, homocysteine, etc.) mean that at least two markers (one plasma-based and one functional) must be included in order to classify individuals correctly [10][11][12]. ...
Background:
Despite being a widely studied concept, the reference interval is the most widely used medical decision-making tool. As such, it is vital that these limits are correctly established and regularly reviewed in the clinical laboratory.
Methods:
The reference population comprised 315 healthy individuals selected a priori from Bizkaia province. Blood and serum samples were sent for subsequent assay of vitamin B12 and folate using three immunochemical methods. Reference values were calculated using non-parametric methods.
Results:
The reference values for serum vitamin B12 and folate were almost identical to those obtained previously using the same methods. Use of new reference values led to an increase in the kappa value despite the low agreement in the case of vitamin B12 (0.4 - 0.62). However, precision obtained for vitamin B12 (94.48 - 96.55%) and folate (95.77 - 97.18%) was very high. The intraclass correlation coefficient ranged from 0.723 to 0.894. Furthermore, a Passing-Bablok regression analysis gave acceptable correlation coefficients of 0.75 - 0.94 for vitamin B12 and 0.92 - 0.95 for folate.
Conclusions:
Vitamin B12 and folate deficiencies are currently being over-diagnosed leading to an increase in the number of unnecessary consultations. The main conclusion that can be drawn from our study has resulted in a change in reference values in our laboratory, with a subsequent increase in our ability to accurately detect possible deficiencies. Furthermore, as this study involved all methods currently in use in the Basque healthcare network, its conclusions can be extrapolated to the whole population covered by Osakidetza, thereby improving the rational use of healthcare funding.
... c o m / l o c a t e / e n v i n t two issues as described in an analytical note released in 2010 (Centers for Disease Control and Prevention, 2010). Several papers also described comparability issues with folate and vitamin B-12 datasets in 1991–1994 and 1999–2006 NHANES surveys (Bock and Eckfeldt, 2011; Yetley and Johnson, 2011; Yetley et al., 2011a Yetley et al., , 2011b Yetley et al., , 2011c). The CDC also reported a purity issue with a perfluorinated compound calibration standard used in their projects since the early 2000s. ...
... The difficulty to evaluate the diagnostic accuracy of B12 testing according to standardized criteria increases the need to assure at least the analytical equivalence of marker results by different assays through the implementation of their traceability to internationally recognized reference materials (RMs) [12,68]. Tracing back the calibration of commercial assays to suitable RMs enables to harmonize patient results and standardize the recommended decision limits [69]. ...
Abstract In our hospital, we are currently working to manage the appropriateness of vitamin B12 (B12) testing. Unfortunately, the classic evidence-based approach is unhelpful in this process and meta-analyzing data on the accuracy of this marker for cobalamin deficiency detection is misleading due to the lack of reference diagnostic methods. The approach currently proposed by the Health Technology Assessment (HTA) enables us to tackle the issue of B12 requests as a "healthcare" problem by considering the position of stakeholders involved in ordering, performing, interpreting the test, and receiving its results. Clinical expectations, methodological issues, and ethical aspects concerning the performance of the test can aid us in providing more guidance on the use of this marker. By building such structured information, hemodialysis patients and pregnant women have emerged as those groups preferentially requiring B12 testing, as it may potentially improve the clinical outcome. To avoid misinterpretation of B12 results more care should be taken in considering its biochemical and biological features, as well as the analytical issues. Spurious values obtained by current automated immunoassays may reflect suboptimal pre-analytical steps as well as known interfering conditions. Furthermore, the harmonization of results by available methods is still a far-reaching goal and the approach to interpret an individual's results should be improved. Tracing a roadmap for B12 testing by exploiting the HTA model to balance the stakeholders' claims and maximizing the patient's outcome may help to manage the marker demand.
... Accurate and precise quantification of blood folate are essential to assess individual folate status and to monitor population changes following folic acid (FA) 5 fortification of foods (1). There is also considerable interest in the association between circulating folate concentrations and variation in genes contributing to folate-mediated one-carbon metabolism, with the homozygous mutation (C677T) of the gene methylenetetrahydrofolate reductase (MTHFR) emerging as a strong modifier of folate status. ...
Standardization of folate measurement is needed for accurate assessment of folate status. We compared the measurement of whole-blood folate by isotope dilution-liquid chromatography-tandem MS (ID-LC-MS/MS) with the historical gold standard microbiological assay (MA) using 3 common calibrators within the frame of the methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism. Seventy-three whole-blood samples with an even distribution of MTHFR C677T genotypes (24 CC, 24 CT, 24 TT) were prepared, and total folate was determined by ID-LC-MS/MS and MA using the following calibrators: 5-methyltetrahydrofolate (5-methylTHF) (Merck), folic acid (FA) (Merck), and FA (Sigma). To compare the methods, 5-formyltetrahydrofolate (5-formylTHF) was excluded in the ID-LC-MS/MS summation of total folate, because it is likely that the majority of 5-formylTHF detected is a pyrazino-s-triazine oxidation product of 5-methylTHF. MA whole-blood folate measured by using the FA calibrators was consistently higher than with the 5-methylTHF calibrator. Differences between dilutions and analysis of spiked whole-blood samples showed a nonlinear response, with overrecovery of 5-methylTHF by ∼23% toward the higher end of the MA calibration range. Significant proportional biases between ID-LC-MS/MS and MA were found in all comparisons except when the MA was calibrated with 5-methylTHF and a higher sample dilution of 1:1600 (regression slope: 1.05; P = 0.31; intercept-21, P = 0.16). Calibration bias and matrix effects in the MA underscore the need for a formally accepted whole-blood folate reference method. ID-LC-MS/MS procedures have the potential to offer a high degree of accuracy; however, further work is needed to determine the origin of the pyrazino-s-triazine derivative.
Background:
Vitamin B12 status is assessed primarily by total serum B12 using competitive binding methods. The lack of availability of a standard material, and high-level reference measurement procedure affects the trueness of B12 results. This results in variation between methods. This study aimed to determine the reference intervals for vitamin B12 on three routine analytical platforms.
Method:
A prospective reference population of healthy individuals was recruited according to the IFCC CRIDL criteria. Vitamin B12 samples were measured on Roche, Beckman and Siemens analytical platforms.
Results:
In total, 300 adult subjects were recruited; the central 95th centile values for B12 for Roche (190-678 ng/ml) and Siemens (181-562 ng/ml) analytical platforms were in a close agreement. Beckman DXi however, showed a significantly lower reference limit (110-562 ng/ml). All reference intervals are in keeping with previously published data but some are not in agreement with manufacturer provided reference interval.
Conclusion:
As the quality of the reference intervals play a significant role in clinical outcome. It is of great importance that laboratories use method-specific reference interval and if possible locally derived reference intervals until further method standardisation occurs.
In cooperation with the Japan Committee for Vitamin Laboratory Standards, the Committee on Nutrition of the Japan Society of Clinical Chemistry has issued a statement on the standardization in Japan of serum folate measurement (sum of 5-methyltetrahydrofolic acid, 5-formyltetrahydrofolic acid, tetrahydrofolic acid, and folic acid). The repeatability and reproducibility-within-laboratory variations presented in the automated analysis (i.e., UniCel, Centaur and Elecsys) confound the diagnosis of hypovitaminosis folate deficiency, and required standardization from clinical and nutritional laboratories. Based on me study conducted by the World Health Organization (WHO), the National Institute of Standards and Technology (NIST) together with Centers for Disease Control and Prevention (CDC) developed a Standard Reference Material (SRM 1955). The SRM 1955 consisted of three levels, the assigned values of which were determined using a liquid chromatography/tandem mass spectrometry (i.e., the reference measurement procedure). The information concentrations of total folate were 2.6 ng/ mL for Level I, 5.8 ng/mL for Level II and 18.0 ng/mL for Level III. Using values of microbiological assay related to the assigned values of SRM 1955 as a comparison value, we investigated the possibility of standardization for the assay values of serum folate as measured by the automated analysis. When the observed values of SRM 1955 and 50 patient sera as measured by automated analysis were compared with the comparison values, the values by UniCel (for levels I and III) and Elecsys (for levels II and III) were plotted within a 95% prediction interval of regression line. The value by Centaur for Level II was plotted outside the 95% prediction interval, while the normalized residuals obtained from three levels of SRM 1955 by three automated analyses were all plotted within ±3.0 intervals. We concluded that SRM 1955 had a physicochemical characterization similar to that of native human serum. In the measurements of 50 patient sera by three automated analyses, we calibrated their observed values for the reference concentration values of SRM 1955. Based on the nutritional criteria classified as deficiency (<3.0 ng/mL), marginal deficiency (3.0-5.9 ng/mL) and low risk (≥ 6.0 ng/ mL), we compared the patients' nutritional status before and after correction with SRM 1955. By calibration for SRM 1955, both the number of patients with marginal deficiency misclassified as deficiency and patients with low risk misclassified as marginal deficiency were decreased. Although the increased number of patients with marginal deficiency misclassified as low risk needed to be further investigated, overall, the decreased number of misclassification verified the usefulness of calibration for SRM 1955. In this study, we used a method-specific calibration procedure for automated analysis, and the observed values were then calibrated for the assigned values of SRM 1955. In the future standardization, we expect all manufacturers to use SRM 1955 in assigning the values of their calibrator product for automated analysis.
p>Los resultados generados por los laboratorios clínicos son elementos claves para el diagnóstico y tratamiento de enfermedades, así como para el seguimiento de los pacientes, por lo tanto se requiere un control metrológico sobre el proceso de medición, el cual determinará el grado de comparabilidad y de confianza requerido sobre los resultados obtenidos. Esta revisión aborda el tema de comparabilidad de las mediciones y de cómo ésta se ve influenciada por factores como la conmutabilidad de los materiales de referencia, los métodos de medición, la disponibilidad de materiales y procedimientos de referencia, además de los intervalos de referencia y límites de decisión para un mensurando en particular. Finalmente se exponen cuáles son los mecanismos adoptados que permitirán garantizar la comparabilidad de las mediciones en esta área.
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This study focuses on an appraisal of the quality, relevance and feasibility of the monitoring of the folate status of (pre)pregnant women. The reason for this study is that such monitoring can support health policy as well as scientific investigations concerning folate intake and e.g. birth defects.
The study comprised the construction of a framework for the appraisal, the collection and study of relevant sources of information and finally the appraisal itself of a variety of folate status monitoring scenario’s, using the framework and the information collected.
Methods to monitor folate status by assessing intake and by assessing biomarkers were evaluated with respect to the type of folate intake measured, the quantitative estimates used, the subgroups studied, the validity of the methods and the way data are collected. Folate intake is mainly assessed by self-report using self-to-fill-in questionnaires. A sufficiently validated instrument for self-reported intake that might be used for long-term and broadly folate status monitoring does not seem to exist. Commonly used biomarkers are red blood cell folate and plasma folate. These markers can be validly assessed , but their relation with exposures or interventions to be monitored is not clear yet.
A variety of possibly relevant monitoring scenario´s was set up by varying six core characteristics: the estimated factor; the subjects measured;the measurement points/periods; the sampling scheme; the level of analysis; the scope . Each scenario defines a specific way of data collection resulting in a specific dataset. The relevance of the scenario is based on the usefulness of that dataset. The usefullness of the datasets is assessed for different purposes, namely for explanatory scientific research, for the development and evaluation of policy to promote folic acid supplement use and for individual patient care.
Three scenario’s are recommended to include in the further analysis. The most relevant one would permanently measure one or more biomarkers of folate status in all pregnancies It is useful for explanatory case-control studies (and cohort and ecologic studies as well) and also for the development and evaluation of FA-supplementation policy. The second most relevant scenario would also make use of biomarkers but only collected in representative samples of e.g. 1 in 100 pregnancies. The datasets could be used for explanatory ecologic studies of pregnancy outcomes as well as for FA-supplementation policy. And the third most relevant scenario would collect data about folic acid supplement intake in the periconceptional period based on retrospective self-reports.These data can be used for ecologic trend studies and for the purposes of FA-supplementation policy.
The feasibility of the implementation of folate monitoring is studied with respect to the fulfillment of some conditions that would precede actual implementation. These conditions are in the domains: quality of the measurement methods; organisational embedment of data collection; sample and data collection competencies; legal and ethical regulations; cost-benefit expectations. No insurmountable hindrances for the implementation of folate intake monitoring through self-report methods seem to exist. The same applies to the implementation of a biomarker monitoring, but the compliance with the requirements dictated by legal regulations might cause a considerable hurdle.
It is finally recommended to proceed with two solutions:
A. folate intake monitoring, either through questionnaires or anonymized biomarker measurements, for the purpose of FA-supplementation policy development and evaluation;
B. non-anonymous (regional) census biomarker monitoring for the purpose of explanatory scientific research of birth defects and pregnancy related disorders.
These solutions serve clearly distinct purposes and could both be implemented via separate paths and in different regions. Solution A is furthermore relatively simple and cost-effective if integrated in FA-supplementation policy. Solution B is technically more complex but feasible if integrated in existing procedures of blood sampling in pregnancy. Future work on these solutions should be directed to the further ascertainment of the feasibility and to the exploration of the commitment of decisive stakeholders.
Acknowledgements
I’d like to thank Hermien de Walle and Marian Bakker from EUROCAT Netherlands, Lolkje de Jong-van den Berg from Department of Pharmacoepidemiology and Pharmaeconomy, RijksUniversiteit Groningen and Henk Blom, Head Metabolic Unit, Clinical Chemistry, VU University Medical Center for reading and commenting earlier draft versions of this report.
Folate status assessments depend primarily on the measurement of biomarkers such as serum and red blood cell folate. Lessons learned from a large national monitoring system such as the National Health and Nutrition Examination Survey and a public health intervention such as the implementation of folic acid fortification in the United States have provided useful insights into the challenges of assessing folate status and possible solutions for addressing these challenges. © 2011 International Union of Biochemistry and Molecular Biology, Inc. 2011.
A roundtable to discuss the measurement of vitamin B-12 (cobalamin) status biomarkers in NHANES took place in July 2010. NHANES stopped measuring vitamin B-12-related biomarkers after 2006. The roundtable reviewed 3 biomarkers of vitamin B-12 status used in past NHANES--serum vitamin B-12, methylmalonic acid (MMA), and total homocysteine (tHcy)--and discussed the potential utility of measuring holotranscobalamin (holoTC) for future NHANES. The roundtable focused on public health considerations and the quality of the measurement procedures and reference methods and materials that past NHANES used or that are available for future NHANES. Roundtable members supported reinstating vitamin B-12 status measures in NHANES. They noted evolving concerns and uncertainties regarding whether subclinical (mild, asymptomatic) vitamin B-12 deficiency is a public health concern. They identified the need for evidence from clinical trials to address causal relations between subclinical vitamin B-12 deficiency and adverse health outcomes as well as appropriate cutoffs for interpreting vitamin B-12-related biomarkers. They agreed that problems with sensitivity and specificity of individual biomarkers underscore the need for including at least one biomarker of circulating vitamin B-12 (serum vitamin B-12 or holoTC) and one functional biomarker (MMA or tHcy) in NHANES. The inclusion of both serum vitamin B-12 and plasma MMA, which have been associated with cognitive dysfunction and anemia in NHANES and in other population-based studies, was preferable to provide continuity with past NHANES. Reliable measurement procedures are available, and National Institute of Standards and Technology reference materials are available or in development for serum vitamin B-12 and MMA.
NHANES measured folate and vitamin B-12 status biomarkers, starting with serum folate from NHANES I (1974-1975) through 2010. Subsequent NHANES measured additional biomarkers [eg, red blood cell folate, serum vitamin B-12, total homocysteine (tHcy), methylmalonic acid, serum folic acid, and 5-methyltetrahydrofolic acid]. Examples of the uses of these data are wide ranging and include public policy applications, the derivation of reference intervals, and research. Periodically, the National Center for Health Statistics and its federal partners convene expert panels to review the use of the folate- and vitamin B-12-related biomarkers in NHANES. These panels have evaluated the need for results to be comparable across time and with published data and the use of crossover studies and adjustment equations to ensure comparability. With the recent availability of reference methods and materials for serum folate and tHcy, NHANES has started to use traceability approaches to enhance the accuracy and comparability of its results. A major user concern over the years has been the use of cutoffs to estimate the prevalence of inadequate folate and vitamin B-12 status. Because these cutoffs depend on the measurement procedure, several expert panels suggested approaches for dealing with cutoff challenges. This review summarizes the history and use of folate- and vitamin B-12-related biomarkers beginning with NHANES I (1974-1975) through 2010.
A roundtable dialogue to discuss "NHANES Monitoring of Biomarkers of Folate and Vitamin B-12 Status" took place in July 2010. This article provides an overview of the meeting and this supplement issue. Although the focus of the roundtable dialogue was on the measurement of folate and vitamin B-12 status biomarkers in NHANES, this article also describes the relevance and importance of these issues for clinical and research laboratories. The roundtable identified the microbiological assay (MA) as the gold standard for measurement of serum and red blood cell folate concentrations. The roundtable noted that differences in results between the Bio-Rad Quantaphase II procedure (Bio-Rad Laboratories, Hercules, CA) that NHANES 1991-1994 and 1999-2006 used and the MA that NHANES 2007-2010 used will require adjustment equations to evaluate time trends. The roundtable found that the close agreement between the serum results for the MA and liquid chromatography-tandem mass spectrometry (LC-MS/MS) procedures supported the conversion to LC-MS/MS for serum folate in future NHANES. The roundtable recognized the uncertainty about whether subclinical vitamin B-12 deficiency is a public health concern but encouraged reinstatement of at least one circulating vitamin B-12 measure and one functional vitamin B-12 status measure in future NHANES. The use of serum vitamin B-12 and plasma methylmalonic acid would provide continuity with past NHANES. The roundtable supported the continued use of the National Institute of Standards and Technology (NIST) reference materials in NHANES biomarker analyses and the further development of additional reference materials by the NIST.
A roundtable to discuss the measurement of folate status biomarkers in NHANES took place in July 2010. NHANES has measured serum folate since 1974 and red blood cell (RBC) folate since 1978 with the use of several different measurement procedures. Data on serum 5-methyltetrahydrofolate (5MTHF) and folic acid (FA) concentrations in persons aged ≥60 y are available in NHANES 1999-2002. The roundtable reviewed data that showed that folate concentrations from the Bio-Rad Quantaphase II procedure (Bio-Rad Laboratories, Hercules, CA; used in NHANES 1991-1994 and NHANES 1999-2006) were, on average, 29% lower for serum and 45% lower for RBC than were those from the microbiological assay (MA), which was used in NHANES 2007-2010. Roundtable experts agreed that these differences required a data adjustment for time-trend analyses. The roundtable reviewed the possible use of an isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) measurement procedure for future NHANES and agreed that the close agreement between the MA and LC-MS/MS results for serum folate supported conversion to the LC-MS/MS procedure. However, for RBC folate, the MA gave 25% higher concentrations than did the LC-MS/MS procedure. The roundtable agreed that the use of the LC-MS/MS procedure to measure RBC folate is premature at this time. The roundtable reviewed the reference materials available or under development at the National Institute of Standards and Technology and recognized the challenges related to, and the scientific need for, these materials. They noted the need for a commutability study for the available reference materials for serum 5MTHF and FA.
A roundtable to discuss the measurement of vitamin B-12 (cobalamin) status biomarkers in NHANES took place in July 2010. NHANES stopped measuring vitamin B-12-related biomarkers after 2006. The roundtable reviewed 3 biomarkers of vitamin B-12 status used in past NHANES--serum vitamin B-12, methylmalonic acid (MMA), and total homocysteine (tHcy)--and discussed the potential utility of measuring holotranscobalamin (holoTC) for future NHANES. The roundtable focused on public health considerations and the quality of the measurement procedures and reference methods and materials that past NHANES used or that are available for future NHANES. Roundtable members supported reinstating vitamin B-12 status measures in NHANES. They noted evolving concerns and uncertainties regarding whether subclinical (mild, asymptomatic) vitamin B-12 deficiency is a public health concern. They identified the need for evidence from clinical trials to address causal relations between subclinical vitamin B-12 deficiency and adverse health outcomes as well as appropriate cutoffs for interpreting vitamin B-12-related biomarkers. They agreed that problems with sensitivity and specificity of individual biomarkers underscore the need for including at least one biomarker of circulating vitamin B-12 (serum vitamin B-12 or holoTC) and one functional biomarker (MMA or tHcy) in NHANES. The inclusion of both serum vitamin B-12 and plasma MMA, which have been associated with cognitive dysfunction and anemia in NHANES and in other population-based studies, was preferable to provide continuity with past NHANES. Reliable measurement procedures are available, and National Institute of Standards and Technology reference materials are available or in development for serum vitamin B-12 and MMA.
A roundtable to discuss the measurement of folate status biomarkers in NHANES took place in July 2010. NHANES has measured serum folate since 1974 and red blood cell (RBC) folate since 1978 with the use of several different measurement procedures. Data on serum 5-methyltetrahydrofolate (5MTHF) and folic acid (FA) concentrations in persons aged ≥60 y are available in NHANES 1999-2002. The roundtable reviewed data that showed that folate concentrations from the Bio-Rad Quantaphase II procedure (Bio-Rad Laboratories, Hercules, CA; used in NHANES 1991-1994 and NHANES 1999-2006) were, on average, 29% lower for serum and 45% lower for RBC than were those from the microbiological assay (MA), which was used in NHANES 2007-2010. Roundtable experts agreed that these differences required a data adjustment for time-trend analyses. The roundtable reviewed the possible use of an isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) measurement procedure for future NHANES and agreed that the close agreement between the MA and LC-MS/MS results for serum folate supported conversion to the LC-MS/MS procedure. However, for RBC folate, the MA gave 25% higher concentrations than did the LC-MS/MS procedure. The roundtable agreed that the use of the LC-MS/MS procedure to measure RBC folate is premature at this time. The roundtable reviewed the reference materials available or under development at the National Institute of Standards and Technology and recognized the challenges related to, and the scientific need for, these materials. They noted the need for a commutability study for the available reference materials for serum 5MTHF and FA.
The aim of the present study was to evaluate standard reference material (SRM) 1955 commutability as a reference material for serum folate using automated methods. We also designed so as to reduce the intermethod variability present in different automated methods.
Using a microbiological assay related to the 'information value' of SRM 1955 as a comparison method, we investigated the possibility of standardization for the assay values of serum folate as measured by the automated methods (Access, Centaur and Elecsys). In the assay of 50 patient sera by these automated methods, we corrected observed values by the SRM 1955 and compared with comparison values.
The observed values of SRM 1955 Levels I, II and III were within or outside (but near) a 95% prediction interval obtained from patient sera by the automated methods. The normalized residuals obtained from SRM 1955 were within ±3.0 (in SD units), which enabled us to conclude that the SRM 1955 had a physicochemical characterization similar to native serum. Twelve patients were assessed as hypofolataemia (<6.0 ng/mL) and 38 patients as normal (≥6.0 ng/mL). Before correction, folate levels in six of 12 patients were lower than 6.0 ng/mL, and those in seven of 38 patients were higher than 6.0 ng/mL with the automated methods. After correction, low levels were found in four of 12 patients, and normal levels were found in 33 of 38 patients.
The use of SRM 1955 would help to reduce the intermethod variability present in different automated methods for serum folate measurement.
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.
Context.—Comparison of different analytical methods in proficiency surveys may be affected by the artificial nature of the survey material.
Objective.—To compare intermethod differences in proficiency survey results between 2 types of survey material, conventional proficiency testing material (PTM) and fresh frozen human serum (FFS), for 3 markers of anemia: ferritin, folate, and vitamin B12.
Design.—Data were gathered from a 2003 survey event in the College of American Pathologists Ligand (“K”) Series, in which the specimens to be tested by each participating laboratory included 1 vial of FFS and 2 vials of PTM with different analyte concentrations. The more than 1600 laboratories subscribing to the survey were not advised as to the nature of the specimens.
Main Outcome Measures.—The bias of each method relative to the median of method means for each analyte and each type of survey material, and the interlaboratory coefficient of variation for each method.
Results.—For each of the 3 analytes, moderate to large method biases were observed. For ferritin, method biases correlated strongly between comparable PTM and FFS specimens (Spearman r = 0.863, P < .001), whereas virtually no correlation was found for folate (r = −0.224, P = .48), and a marginally significant correlation existed for B12 (r = 0.55, P = .049).
Conclusions.—With ferritin, proficiency survey performance of PTM is similar to that of FFS, implying that method biases relate mainly to calibration. With folate and to a lesser extent with B12, PTM and FFS exhibit different method biases, implying that the biases reflect analyte heterogeneity and/or matrix effects.
NHANES measured folate and vitamin B-12 status biomarkers, starting with serum folate from NHANES I (1974-1975) through 2010. Subsequent NHANES measured additional biomarkers [eg, red blood cell folate, serum vitamin B-12, total homocysteine (tHcy), methylmalonic acid, serum folic acid, and 5-methyltetrahydrofolic acid]. Examples of the uses of these data are wide ranging and include public policy applications, the derivation of reference intervals, and research. Periodically, the National Center for Health Statistics and its federal partners convene expert panels to review the use of the folate- and vitamin B-12-related biomarkers in NHANES. These panels have evaluated the need for results to be comparable across time and with published data and the use of crossover studies and adjustment equations to ensure comparability. With the recent availability of reference methods and materials for serum folate and tHcy, NHANES has started to use traceability approaches to enhance the accuracy and comparability of its results. A major user concern over the years has been the use of cutoffs to estimate the prevalence of inadequate folate and vitamin B-12 status. Because these cutoffs depend on the measurement procedure, several expert panels suggested approaches for dealing with cutoff challenges. This review summarizes the history and use of folate- and vitamin B-12-related biomarkers beginning with NHANES I (1974-1975) through 2010.
Anaemia is a major global health problem. Although the main cause is iron deficiency, anaemia also results from other nutritional deficiencies (folate and vitamin B12), haemolytic disorders including haemoglobinopathies, and bone marrow disorders. Accurate diagnosis of anaemia is dependent on reliable diagnostic tests and reference ranges, which in turn are dependent on effective standardisation. Standardisation is achieved through the availability of reference materials and reference measurement procedures. International biological reference materials have therefore been developed to standardise and control diagnostic tests for anaemia for a diverse range of analytes including total haemoglobin and haemoglobin types, ferritin, the serum transferrin receptor, serum vitamin B12 and folate, whole blood folate, and alloantibodies which mediate immune haemolytic anaemia.
Unambiguous and consistent concepts and terms such as measurand, metrological traceability, measurement uncertainty, comparability of measurement results, target measurement uncertainty, etc., must govern the description of measurements in order to enable a valid comparison of measurement results. That is not yet the case as numerous workshops over the last decade have shown worldwide and as chemical literature continuously displays. For international trade in food and feed to be fair, for border-crossing implementation of environmental regulations to be the same for all parties concerned, for interchangeability of results of clinical measurements to become a reality, for any border-crossing interpretation of measurement results in chemistry to become possible, well understood and mutually accepted, common and well defined concepts and terms are essential. Similarly, their translations from one language--English--to 30-40 other languages, must be realized and fixed unequivocally. Countries using English as common language have not yet fully realized that they are at a considerable advantage over countries where such translated terms describing concepts may not yet be available, let alone understood and accepted. A number of ambiguities in the definitions and terms are described which illustrate the importance of the revision (1997-2007) of the International Vocabulary of Metrology (VIM), henceforth conveniently called "VIM3", especially since chemical measurement is covered in this VIM for the first time in history: 'measurand' 'metrological comparability of measurement results' 'metrology' 'metrological compatibility of measurement results' 'measurement result' 'metrological traceability' (incl 'to the SI') 'measurement uncertainty' 'target measurement uncertainty' 'calibration hierarchy' 'quantity' and many others. It is concluded that the revised VIM is of primordial importance for good understanding within and between the measurement communities worldwide.
In patient and population samples, generation of analytical results that are comparable and independent of the measurement system, time, and location is essential for the utility of laboratory information supplied in healthcare. Obtaining analytical measurement results with such characteristics is the aim of traceability in laboratory medicine. As awareness of the benefits of having traceable measurement results has increased, associated efforts have been directed toward making traceability a regulatory requirement and developing approaches to enable and facilitate the implementation of traceability. Although traceability has been a main focus of many laboratory standardization activities in the past, discussions are still ongoing with regard to traceability and its implementation.
This review provides information about the traceability concept and what needs can be fulfilled and benefits achieved by the availability of traceable measurement results. Special emphasis is given to the new metrological terminology introduced with this concept. The review addresses and describes approaches for technical implementation of traceable methods as well as the associated challenges. Traceability is also discussed in the context of other activities to improve the overall measurement process.
Establishing metrological traceability of measurement results satisfies basic clinical and public health needs, thus improving patient care and disease control and prevention. Large advances have been made to facilitate the implementation of traceability. However, details in the implementation process, such as lack of available commutable reference materials and insufficient resources to develop new reference measurement systems continue to challenge the laboratory medicine community.
Proficiency testing using stabilized control materials has been used for decades as a means of monitoring and improving performance in the clinical laboratory. Often, the commonly used proficiency testing materials exhibit "matrix effects" that cause them to behave differently from fresh human specimens in certain clinical analytic systems. Because proficiency testing is the primary method in which regulatory agencies have chosen to evaluate clinical laboratory performance, the College of American Pathologists (CAP) has proposed guidelines for investigating the influence of matrix effects on their Survey results. The purpose of this investigation was to determine the feasibility, usefulness, and potential problems associated with this CAP Matrix Effect Analytical Protocol, in which fresh patient specimens and CAP proficiency specimens are analyzed simultaneously by a field method and a definitive, reference, or other comparative method. The optimal outcome would be that both the fresh human and CAP Survey specimens agree closely with the comparative method result. However, this was not always the case. Using several different analytic configurations, we were able to demonstrate matrix and calibration biases for several of the analytes investigated.
Comparison of different analytical methods in proficiency surveys may be affected by the artificial nature of the survey material.
To compare intermethod differences in proficiency survey results between 2 types of survey material, conventional proficiency testing material (PTM) and fresh frozen human serum (FFS), for 3 markers of anemia: ferritin, folate, and vitamin B12.
Data were gathered from a 2003 survey event in the College of American Pathologists Ligand ("K") Series, in which the specimens to be tested by each participating laboratory included 1 vial of FFS and 2 vials of PTM with different analyte concentrations. The more than 1600 laboratories subscribing to the survey were not advised as to the nature of the specimens.
The bias of each method relative to the median of method means for each analyte and each type of survey material, and the interlaboratory coefficient of variation for each method.
For each of the 3 analytes, moderate to large method biases were observed. For ferritin, method biases correlated strongly between comparable PTM and FFS specimens (Spearman r = 0.863, P < .001), whereas virtually no correlation was found for folate (r = -0.224, P = .48), and a marginally significant correlation existed for B12 (r = 0.55, P = .049).
With ferritin, proficiency survey performance of PTM is similar to that of FFS, implying that method biases relate mainly to calibration. With folate and to a lesser extent with B12, PTM and FFS exhibit different method biases, implying that the biases reflect analyte heterogeneity and/or matrix effects.
Total homocysteine (tHCY) and folate are interrelated biomarkers for arteriosclerosis and coronary heart disease. Although many different methods for both tHCY and folate are clinically available, the intermethod and interlaboratory results are often poor, resulting in the need for a matrix reference material and reference methods. The National Institute of Standards and Technology (NIST) has developed isotope dilution liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/ tandem mass spectrometry (LC/MS/MS) methods for determination of tHCY and several folate forms including 5-methyltetrahydrofolic acid (5MT) and folic acid (FA). Additionally, a method for simultaneous measurement of tHCY, 5MT, and FA has been developed and validated. In collaboration with the Centers for Disease Control and Prevention (CDC), mass spectrometric methods and methods used in clinical laboratories have been applied to characterize a new Standard Reference Material (SRM), SRM 1955, "Homocysteine and Folate in Human Serum," containing low, medium, and high levels of tHCY and 5MT. Additionally, FA, 5-formyltetrahydrofolic acid (5FT), vitamin B12, and total folate values are provided. Use of the new SRM should improve clinical measurements and will permit traceability to internationally recognized certified reference materials, as described by European Directive 98/79/EC on in vitro diagnostic medical devices.
Current clinical methods for folate give different results and cannot measure the various forms of folate. We developed an isotope-dilution tandem mass spectrometric method coupled to liquid chromatography (LC/MS/MS) as a candidate reference method for 5-methyltetrahydrofolic acid (5MeTHF), 5-formyltetrahydrofolic acid (5FoTHF), and folic acid (FA) in human serum.
We quantitatively isolated folates from 275 microL of serum with a phenyl solid-phase extraction cartridge, then detected and quantified them in stabilized serum extracts by positive-ion electrospray ionization LC/MS/MS. We used an isocratic mobile phase of acetic acid in organic solvent on a C(8) analytical column. (13)C-labeled folates were used as internal standards.
Limits of detection in serum were 0.13 (5MeTHF), 0.05 (5FoTHF), and 0.07 (FA) nmol/L. Within- and between-run imprecision (CV) was <7% for 5MeTHF and <10% for 5FoTHF at concentrations >0.5 nmol/L, and <10% for FA at concentrations >2.0 nmol/L. Total folate (TFOL) concentrations determined by competitive protein binding radioassay were approximately 9% lower than results obtained with LC/MS/MS. The microbiologic assay gave approximately 15% higher TFOL results with FA calibrator and no difference with 5MeTHF calibrator. The mean (SD) [range] TFOL in 42 sera was 35.5 (17.8) [6.5-75.6] nmol/L. Thirty-two samples with TFOL <50 nmol/L had, on average, 93.3% 5MeTHF, 2.3% FA, and 4.4% 5FoTHF. Ten samples with TFOL >50 nmol/L had, on average, 81.7% 5MeTHF, 15.7% FA, and 2.5% 5FoTHF.
This stable-isotope-dilution LC/MS/MS method can quantify 5MeTHF, 5FoTHF, and FA in serum. Currently used clinical assays agree with this candidate reference method.
Maintaining accurate laboratory measurements over time is crucial for assuring appropriate patient care and disease management. Accurate results over time and location are achieved by standardising measurements and establishing traceability to a reference system. Reference materials are key components of such reference systems and for establishing traceability. Commutability of reference materials is a critical property to ensure they are fit for use. Commutability is defined as the equivalence of the mathematical relationships between the results of different measurement procedures for a reference material and for representative samples from healthy and diseased individuals. This material characteristic is of special importance for measurement procedures that are optimised for measuring analytes directly in patient samples. The commutability of a reference material is measurement procedure specific and its assessment requires special experimental designs. This review explains the importance of commutability and summarises different experimental approaches described in the literature that have been used to assess the commutability of reference materials in clinical chemistry.
Biomarkers of folate status in NHANES: a roundtable summary): nn–nn. 2. Yetley EA, Johnson CL. Folate and vitamin B-12 biomarkers in NHANES: history of their measurement and use
- Ea Yetley
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Yetley EA, Pfeiffer CM, Phinney KW, et al. Biomarkers of folate status in NHANES: a roundtable summary. Am J Clin Nutr 2011;94(suppl): nn–nn. 2. Yetley EA, Johnson CL. Folate and vitamin B-12 biomarkers in NHANES: history of their measurement and use. Am J Clin Nutr 2011; 94(suppl):nn–nn.
International vocabulary of metrology: basic and general concepts and associated terms (VIM). Sèvres, France: JCGM, 2008
- Joint Committee for Guides in Metrology (JCGM)
International Standard ISO 17511 In vitro diagnostic medical devices—measurement of quantities in biological samples—metrological traceability of values assigned to calibrators and control materials
International Organization for Standardization (ISO). International
Standard ISO 17511. In vitro diagnostic medical devices—measurement
of quantities in biological samples—metrological traceability of values
assigned to calibrators and control materials. Geneva, Switzerland: ISO,
2003.