Linda M. Thienpont

Vitamin D status as an international issue: National surveys and the problem of standardization

Abstract Wide spread variation in measurement results of total 25-hydroxyvitamin D (25(OH)D) confounds international efforts to develop evidence-based clinical guidelines. Accordingly, NIH Office of Dietary Supplements (ODS) in collaboration with CDC National Center for Environmental Health (NCEH), National Institute of Standards and Technology (NIST) and Ghent University established the Vitamin D Standardization Program (VDSP) in November 2010. VDSP objectives include: (1) standardize 25(OH)D concentration measurements in national health surveys around the world, (2) evaluate survey differences, (3) extend standardization efforts to assay manufacturers, and to clinical, commercial, and research laboratories, (4) promote standardization of emerging metabolites of vitamin D status, and (5) enable the use of standardized data in patient care and public health. An interlaboratory comparison study is being conducted to assess measurement variability among current assays. Participants include national health surveys from Australia, Canada, Germany, Ireland, Mexico, South Korea, UK and USA, 15 assay manufacturers, and two external quality assurance programs. CDC will implement a formal laboratory certification program. Standardization activities will use single-donor, fresh-frozen serum collected using the CLSI C37 protocol. Initial assay performance criteria, based on biological variability data, are ≤10 % imprecision and ≤5 % bias in relation to the reference values. An ancillary study on commutability of NIST SRM 972a, external quality assurance testing materials is included. To increase the comparability of existing data from different national surveys, master equations will be developed to facilitate the conversion of already existing national survey data to the NIST-Ghent University reference measurement procedures.

Roadmap for Harmonization of Clinical Laboratory Measurement Procedures

Results between different clinical laboratory measurement procedures (CLMP) should be equivalent, within clinically meaningful limits, to enable optimal use of clinical guidelines for disease diagnosis and patient management. When laboratory test results are neither standardized nor harmonized, a different numeric result may be obtained for the same clinical sample. Unfortunately, some guidelines are based on test results from a specific laboratory measurement procedure without consideration of the possibility or likelihood of differences between various procedures. When this happens, aggregation of data from different clinical research investigations and development of appropriate clinical practice guidelines will be flawed. A lack of recognition that results are neither standardized nor harmonized may lead to erroneous clinical, financial, regulatory, or technical decisions. Standardization of CLMPs has been accomplished for several measurands for which primary (pure substance) reference materials exist and/or reference measurement procedures (RMPs) have been developed. However, the harmonization of clinical laboratory procedures for measurands that do not have RMPs has been problematic owing to inadequate definition of the measurand, inadequate analytical specificity for the measurand, inadequate attention to the commutability of reference materials, and lack of a systematic approach for harmonization. To address these problems, an infrastructure must be developed to enable a systematic approach for identification and prioritization of measurands to be harmonized on the basis of clinical importance and technical feasibility, and for management of the technical implementation of a harmonization process for a specific measurand.

Traceability in Laboratory Medicine

BACKGROUNDnIn 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.nnnCONTENTnThis 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.nnnSUMMARYnEstablishing 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.

Report of the IFCC Working Group for Standardization of Thyroid Function Tests; Part 1: Thyroid-Stimulating Hormone

BACKGROUNDnLaboratory testing of serum thyroid-stimulating hormone (TSH) is an essential tool for the diagnosis and management of various thyroid disorders whose collective prevalence lies between 4% and 8%. However, between-assay discrepancies in TSH results limit the application of clinical practice guidelines.nnnMETHODSnWe performed a method comparison study with 40 sera to assess the result comparability and performance attributes of 16 immunoassays.nnnRESULTSnThirteen of 16 assays gave mean results within 10% of the overall mean. The difference between the most extreme means was 39%. Assay-specific biases could be eliminated by recalibration to the overall mean. After recalibration of singlicate results, all assays showed results within the biological total error goal (22.8%), except for 1 result in each of 4 assays. For a sample with a TSH concentration of 0.016 mIU/L, 6 assays either did not report results or demonstrated CVs >20%. Within-run and total imprecision ranged from 1.5% to 5.5% and 2.5% to 7.7%, respectively. Most assays were able to match the internal QC targets within 5%. Within-run drifts and shifts were observed.nnnCONCLUSIONSnHarmonization of TSH measurements would be particularly beneficial for 3 of the 16 examined assays. These data demonstrate that harmonization may be accomplished by establishing calibration traceability to the overall mean values for a panel of patient samples. However, the full impact of the approach must be further explored with a wider range of samples. Although a majority of assays showed excellent quality of performance, some would benefit from improved within-run stability.

Report of the IFCC Working Group for Standardization of Thyroid Function Tests; Part 2: Free Thyroxine and Free Triiodothyronine

BACKGROUNDnFree thyroxine (FT4) and free triiodothyronine (FT3) measurements are useful in the diagnosis and treatment of a variety of thyroid disorders. The IFCC Scientific Division established a Working Group to resolve issues of method performance to meet clinical requirements.nnnMETHODSnWe compared results for measurement of a panel of single donor sera using clinical laboratory procedures based on equilibrium dialysis-isotope dilution-mass spectrometry (ED-ID-MS) (2 for FT4, 1 for FT3) and immunoassays from 9 manufacturers (15 for FT4, 13 for FT3) to a candidate international conventional reference measurement procedure (cRMP) also based on ED-ID-MS.nnnRESULTSnFor FT4 (FT3), the mean bias of 2 (4) assays was within 10% of the cRMP, whereas for 15 (9) assays, negative biases up to -42% (-30%) were seen; 1 FT3 assay was positively biased by +22%. Recalibration to the cRMP eliminated assay-specific biases; however, sample-related effects remained, as judged from difference plots with biologic total error limits. Correlation coefficients to the cRMPs ranged for FT4 (FT3) from 0.92 to 0.78 (0.88 to 0.30). Within-run and total imprecision ranged for FT4 (FT3) from 1.0% to 11.1% (1.8% to 9.4%) and 1.5% to 14.1% (2.4% to 10.0%), respectively. Approximately half of the manufacturers matched the internal QC targets within approximately 5%; however, within-run instability was observed.nnnCONCLUSIONSnThe study showed that most assays had bias largely correctable by establishing calibration traceability to a cRMP and that the majority performed well. Some assays, however, would benefit from improved precision, within-run stability, and between-run consistency.

Standardization of measurements of 25-Hydroxyvitamin D3 and D2

Abstract The vitamin D status is increasingly assessed/monitored in different populations, research cohorts and individual patients. This is done by measuring the liver metabolites 25-hydroxyvitamin D3 and D2 as biomarkers. Recommendations for using specific serum concentrations of these biomarkers to assess a persons vitamin D status were done. This requires current vitamin D assays to be sufficiently accurate over time, location and laboratory procedures. In view of the fact that several studies demonstrated that current 25(OH)D measurement methods do not meet this prerequisite, standardization is needed. This paper rehearses the basic concept of standardization, in particular applied to measurements of 25-hydroxyvitamin D. Progress has been made by establishing a reference measurement system consisting of reference methods and reference materials. Coordinated efforts to improve the accuracy and standardize measurements are being performed by organizations such as the U.S. NIH, the CDC and Prevention, the NIST together with their national and international partners. Beyond describing the available reference measurement system and its use as calibration hierarchy to establish traceability of measurements with routine laboratory methods to the SI-unit, this report will also focus on other aspects considered essential for a successful and sustainable standardization, such as analytical issues related to the definition of the measurand and analytical performance goals.

Trueness verification in European external quality assessment schemes: time to care about the quality of the samples

An external quality assessment (EQA) survey on 14 fresh‐frozen, single‐donation sera assigned with reference measurement procedure (RMP) values revealed a mean bias of +5.2% and +3.7% for the cholesterol oxidase and the photometric glucose oxidase procedure groups, respectively. Conversely, on lyophilized sera, the same procedure groups showed almost bias‐free results, the differences from the RMP values being only −0.8% for cholesterol and +0.7% for glucose. These data, which are in fairly good agreement with the literature, suggest the existence of artificial matrix effects in processed materials. Therefore they indicate that, currently, assessment of trueness is hampered in many European EQA schemes, as most of them use lyophilized sera. This approach may give a false impression about the trueness of laboratory results as well as carrying the risk that laboratories calibrated on the RMP values of the survey samples could make errors in patient testing. Consequently, if European EQA is willing to fulfil a post‐market vigilance function of the performance of in vitro diagnostic medical devices, then the time has come to tackle the problem of the quality of the survey samples. EQA organizers urgently need to make an effort to seek out materials that analytically behave like authentic clinical specimens. In the meantime, alternative approaches should be used. Although not ideal, the special survey described in this article is one of the possibilities. Naturally, it implies logistic problems and increased costs for the individual EQA schemes. However, both can be overcome with the cooperation of the predominantly nationally organized schemes.

A Progress Report of the IFCC Committee for Standardization of Thyroid Function Tests

Background: The IFCC Committee for Standardization of Thyroid Function Tests aims at equivalence of laboratory test results for free thyroxine (FT4) and thyrotropin (TSH). Objectives: This report describes the phase III method comparison study with clinical samples representing a broad spectrum of thyroid disease. The objective was to expand the feasibility work and explore the impact of standardization/harmonization in the clinically relevant concentration range. Methods: Two sets of serum samples (74 for FT4, 94 for TSH) were obtained in a clinical setting. Eight manufacturers participated in the study (with 13 FT4 and 14 TSH assays). Targets for FT4 were set by the international conventional reference measurement procedure of the IFCC; those for TSH were based on the all-procedure trimmed mean. The manufacturers recalibrated their assays against these targets. Results: All FT4 assays were negatively biased in the mid- to high concentration range, with a maximum interassay discrepancy of approximately 30%. However, in the low range, the maximum deviation was approximately 90%. For TSH, interassay comparability was reasonable in the mid-concentration range, but worse in the pathophysiological ranges. Recalibration was able to eliminate the interassay differences, so that the remaining dispersion of the data was nearly entirely due to within-assay random error components. The impact of recalibration on the numerical results was particularly high for FT4. Conclusions: Standardization and harmonization of FT4 and TSH measurements is feasible from a technical point of view. Because of the impact on the numerical values, the implementation needs careful preparation with the stakeholders.

Harmonization of Serum Thyroid-Stimulating Hormone Measurements Paves the Way for the Adoption of a More Uniform Reference Interval

BACKGROUNDnThe IFCC Committee for Standardization of Thyroid Function Tests developed a global harmonization approach for thyroid-stimulating hormone measurements. It is based on a multiassay method comparison study with clinical serum samples and target setting with a robust factor analysis method. Here we describe the Phase IV method comparison and reference interval (RI) studies conducted with the objective to recalibrate the participating assays and demonstrate the proof-of-concept.nnnMETHODSnFourteen manufacturers measured the harmonization and RI panel; 4 of them quantified the harmonization and first follow-up panel in parallel. All recalibrated their assays to the statistically inferred targets. For validation, we used desirable specifications from the biological variation for the bias and total error (TE). The RI measurements were done with the assays current calibrators, but data were also reported after transformation to the new calibration status. We estimated the pre- and postrecalibration RIs with a nonparametric bootstrap procedure.nnnRESULTSnAfter recalibration, 14 of 15 assays met the bias specification with 95% confidence; 8 assays complied with the TE specification. The CV of the assay means for the harmonization panel was reduced from 9.5% to 4.2%. The RI study showed improved uniformity after recalibration: the ranges (i.e., maximum differences) exhibited by the assay-specific 2.5th, 50th, and 97.5th percentile estimates were reduced from 0.27, 0.89, and 2.13 mIU/L to 0.12, 0.29, and 0.77 mIU/L.nnnCONCLUSIONSnWe showed that harmonization increased the agreement of results from the participating immunoassays, and may allow them to adopt a more uniform RI in the future.

Assessment of the state-of-the-art trueness and precision of serum total-calcium and glucose measurements in Finnish laboratories - the QSL-Finland study

The purpose of the QSL-Finland study was to assess the state-of-the-art trueness and precision of serum total-calcium and glucose measurements in Finnish clinical laboratories. For this purpose, 21 hospitals and clinical institutes were selected. They measured six single donation sera, the total-calcium (t-calcium) and glucose content of which had been determined by ion chromatography and isotope dilution-gas chromatography-mass spectrometry (ID-GC-MS) reference methods. The results were interpreted in light of specifications for imprecision, bias and total error of routine methods that have been proposed in the past. The data revealed that the performance of t-calcium and glucose methods is generally acceptable in Finnish clinical laboratories. This study did not lead to a separation of laboratories according to accreditation. In consequence, it seems that accreditation, in its present form, cannot substitute dedicated quality assurance practices.