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Background: We developed and evaluated a candidate reference measurement procedure (RMP) to standardize testosterone measurements, provide highly accurate and precise value assignments for the CDC Hormone Standardization Program, and ensure accurate and comparable results across testing systems and laboratories., Methods: After 2 liquid/liquid extractions of serum with a combination of ethyl acetate and hexane, we quantified testosterone by isotope-dilution liquid chromatography-tandem mass spectrometry with electrospray ionization in the positive ion mode monitoring 289→97 m/z (testosterone) and 292→112 m/z ((3)C(13) testosterone). We used calibrator bracketing and gravimetric measurements to give higher specificity and accuracy to serum value assignments. The candidate RMP was evaluated for accuracy by use of NIST-certified reference material SRM971 and validated by split-sample comparison to established RMPs. We evaluated intraassay and interassay imprecision, measurement uncertainty, potential interferences, and matrix effects., Results: A weighted Deming regression comparison of the candidate RMP to established RMPs showed agreement with no statistical difference (slope 0.99, 95% CI 0.98-1.00, intercept 0.54, 95% CI -1.24 to 2.32) and a bias of ≤0.3% for NIST SRM971. The candidate RMP gave maximum intraassay, interassay, and total percent CVs of 1.5%, 1.4%, and 1.7% across the concentrations of testosterone typically found in healthy men and women. We tested structural analogs of testosterone and 125 serum samples and found no interferences with the measurement., Conclusions: This RMP for testosterone can serve as a higher-order standard for measurement traceability and can be used to provide an accuracy base to which routine methods can be compared in the CDC Hormone Standardization Program., (© 2012 American Association for Clinical Chemistry)

The IFCC Working Group for Standardization of Thyroid Function Tests proposes a candidate international conventional reference procedure (RMP) for measurement of the amount-of-substance concentration of free thyroxine in plasma/serum at physiological pH 7.40 and temperature (37.0°C). The unit for reporting measurement results is, by convention, pmol/L. The RMP is based on equilibrium dialysis isotope dilution-liquid chromatography/tandem mass spectrometry (ED-ID-LC/tandem MS). The rationale for proposing a conventional RMP is that, because of the physical separation step, it is unknown whether the measurement truly reflects the concentration of free thyroxine (FT4) in serum. Therefore, the ED part of the RMP has to strictly adhere to the following conditions: use of a dialysis buffer with a biochemical composition resembling the ionic environment of serum/plasma as closely as possible; buffering of the sample to a pH of 7.40 (at 37.0°C) before dialysis, however, without additional dilution; dialysis in a device with a dialysand/dialysate compartment of identical volume and separated by a membrane of regenerated cellulose and adequate cut-off; thermostatic control of the temperature during dialysis at 37.0°C±0.50°C. The convention does not apply to the ID-LC/tandem MS part, provided it is eligible to be nominated for review by the Joint Committee for Traceability in Laboratory Medicine. Here, we describe the ED procedure, inclusive its validation and transferability, in greater detail. We recommend a protocol for successful calibration, measurement and monitoring of the accuracy/trueness and precision of the candidate conventional RMP. For details on our ID-LC/tandem MS procedures, we refer to the Supplement.

Background: 25-hydroxyvitamin D [25(OH)D] assays are characterized by poor between-assay comparability. This result emphasizes the need for reference measurement procedures (RMPs) to establish calibration traceability and assist in method validation. We aimed at developing candidate RMPs on the basis of isotope- dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) for separate quantification of serum 25(OH)D2 and 25(OH)D3., Methods: Hexa-deuterated 25(OH)D3/D2 was added to serum. This mixture was extracted with n-hexane and fractionated on Sephadex LH-20 before 2-dimensional LC-MS/MS. In the first dimension, both procedures used a C4 column; however, in the second dimension, the 25(OH)D2 procedure used a C18 and the 25(OH)D3 procedure used a Zorbax SB-CN column. Calibration was traceable to the NIST Standard Reference Material (SRM) 2972. Validation comprised assessment of interference and limit of quantification/detection. Imprecision and trueness were validated by analysis of the SRM 972 against specifications (CV<5% and bias<1.7%). The expanded uncertainty for quadruplicate measurements was estimated., Results: Testing of potentially interfering substances was negative. Interference by 3-epi-25(OH)D3 was resolved by sufficient chromatographic resolution. The limits of quantification/detection were 1.1 nmol/L and 0.09 pmol/L for 25(OH)D3 and 1.2 nmol/L and 0.05 pmol/L for 25(OH)D(2). Mean total CVs and differences from the SRM 972 target (±1-sided 95% CI) were 2.1% and 1.1%±1.5% [25(OH)D3] and 3% and 1.3%±0.6% [25(OH)D2], respectively. The respective expanded uncertainties were 3.4% and 3.9%., Conclusions: From the validation data, we conclude that we achieved our objective of 2 state-of-the-art candidate RMPs for serum 25(OH)D3 and 25(OH)D2.

Background: Laboratory testing is an essential tool for diagnosis and management of thyroid diseases. However, the current status of standardization hampers the interchangeability of results. To improve this situation, the Working Group for Standardization of Thyroid Function Tests was established., Methods: Method comparisons were organized for measurement of human thyroid stimulating hormone (TSH), and free and total thyroid hormone in serum from apparently healthy donors. The aim was to assess the status of standardization and the quality of the performance of current routine assays. A second objective was to investigate the effect of mathematical recalibration of the results using their relationship to the overall mean (TSH) or the reference measurement procedure values (other thyroid hormones)., Results: The need for standardization was shown to be highest for free thyroid hormone and total triiodothyronine measurements, while the majority of TSH and total thyroxine assays agreed within 10% of the reference. Most assays showed good performance. However, some could benefit from improved precision, consistency of calibration, or within- and between-run stability. Recalibration eliminated assay-specific bias. Thus, the residual spread was due to within-method effects. Not withstanding, sample-related effects remained., Conclusions: These studies confirmed the feasibility of standardization based on method comparison with native sera, but highlighted the need to resolve issues, such as sample-related effects. In view of the fact that in this phase the project worked with samples from individuals with euthyroid status, the next method comparison shall place emphasis on challenging the performance of the assays with clinical samples and expanding the covered measurement range.

The Publisher regrets that this article is an accidental duplication of an article that has already been published, doi:10.1016/j.cca.2010.09.032. The duplicate article has therefore been withdrawn., (Copyright © 2010. Published by Elsevier B.V. All rights reserved.)

Background: Serum free thyroxine (FT4) testing in pregnancy is known to be challenging for immunoassays (IAs). We verified the reliability of FT4 results by 3 commercial IAs throughout pregnancy, by comparison of the pattern to that obtained with an equilibrium dialysis isotope dilution-mass spectrometry (ED ID-MS) candidate reference measurement procedure., Methods: Pregnant females (107) and age-matched non-pregnant controls (26) were enrolled. The IAs tested were those performed on the Cobas 6000 (Roche Diagnostics), ARCHITECT i2000SR (Abbott Diagnostics) and Immulite 2000 (Siemens Healthcare) platforms., Results: Compared to the controls (mean FT4: 18.2pmol/L), ED ID-MS gave in the late first trimester pregnancy an 8.8% lower (p<0.05) mean; in the second trimester it was 29.1% lower (p<0.001), to stabilize in the third trimester (p=0.99). Similar observations were made for the Cobas and Immulite IAs. The ARCHITECT IA showed no significant decrease in the late first trimester (mean 13.5pmol/L versus 13.6pmol/L in controls), but a significant, less pronounced, decrease in the second and third trimesters (15% and 14.4%, respectively). All IAs were susceptible towards alterations in T4 binding proteins during pregnancy., Conclusion: We proved that IAs may give a FT4 pattern during pregnancy similar to that obtained by ED ID-MS., (Copyright 2010 Elsevier B.V. All rights reserved.)

Background: Because total thyroid hormone testing is performed on many automated clinical chemistry instruments, the IFCC Scientific Division commissioned the Working Group for Standardization of Thyroid Function Tests to include total thyroxine (TT4) and total triiodothyronine (TT3) in its standardization efforts., Methods: Existing SI-traceable reference measurement procedures (RMPs) were used to assign TT4 and TT3 values to 40 single-donor serum samples for subsequent use in a method comparison study with 11 TT4 and 12 TT3 immunoassays. Data from comparison of each immunoassay with the RMPs provided a basis for mathematical assay recalibration., Results: Seven TT4 assays had a mean bias within 10% of the RMP, but 2 deviated by an average of -12% and another 2 by +17%. All TT3 assays showed positive biases, 4 within and 8 outside 10%, up to 32%. Mathematical recalibration effectively eliminated assay-specific biases, but sample-related effects remained, particularly for TT3. Correlation coefficients with the RMPs ranged from 0.82 to 0.97 for TT4 and from 0.32 to 0.92 for TT3. The within-run and total imprecision ranges for TT4 were 1.4% to 9.1% and 3.0% to 9.4%, respectively, and for TT3 2.1% to 7.8% and 2.8% to 12.7%, respectively. Approximately one-half of the assays matched the internal QC targets within approximately 5%; however, we observed within-run drifts/shifts., Conclusions: The study showed that of the assays we examined, only 4 TT4 but the majority of the TT3 assays needed establishment of calibration traceability to the existing RMPs. Most assays performed well, but some would benefit from improved precision, within-run stability, and between-run consistency.

Background: Laboratory 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., Methods: We performed a method comparison study with 40 sera to assess the result comparability and performance attributes of 16 immunoassays., Results: Thirteen 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., Conclusions: Harmonization 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.

Background: Free 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., Methods: We 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., Results: For 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., Conclusions: The 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.

Background: Measurement of circulating insulin may improve the classification and management of diabetes mellitus and assist in treating people with insulin resistance., Methods: A work group convened by the American Diabetes Association evaluated results for a panel of 39 single donor sera measured by 10 commercial insulin methods from 9 manufacturers against an isotope dilution-liquid chromatography/tandem mass spectrometry (IDMS) measurement procedure calibrated using purified recombinant insulin. We used a candidate primary (pure substance) reference material, pooled serum, and single donor sera to evaluate approaches to achieve improved agreement of results between the routine and reference measurement procedures., Results: Four of 10 methods had >or=95% of individual serum results within 32% of the IDMS concentrations. However, the bias vs IDMS was more than 15.5% for 7 of 10 methods in 36%-100% of individual samples. A purified recombinant insulin preparation used as a common calibrator did not improve harmonization of results among routine methods but was not used as instructed by all participants. Calibration using serum pools achieved bias <15.5% for nearly all results in the concentration range covered by the pools (>60 pmol/L). Calibration using a panel of individual sera was the most effective to improve harmonization of results over the full measuring range., Conclusions: Agreement among methods can be improved by establishing traceability to the IDMS procedure using a panel of native sera. Pooled sera may be useful as trueness control materials. The usefulness of the pure insulin primary reference material [candidate reference material for insulin (cRMI)] requires clarification of protocols used by manufacturers.

Background: The recent interest of clinical laboratories in developing serum testosterone assays based on isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) stems from the lack of accuracy of direct immunoassays. In this study, we assessed the accuracy and state of standardization (traceability) of 4 published ID-LC-MS/MS procedures in a method comparison with an ID-gas chromatography (GC)-MS reference measurement procedure listed in the database of the Joint Committee for Traceability in Laboratory Medicine., Methods: The study used 58 specimens from different patient categories. Each specimen was measured in triplicate (ID-LC-MS/MS) and quadruplicate (ID-GC-MS) in independent runs., Results: The testosterone concentrations by ID-GC-MS were 0.2-4.4 nmol/L (women), 0.2-2.0 nmol/L (hypogonadal man), and 10.1-31.3 nmol/L (normogonadal men). For ID-GC-MS, the CV was nearly constant, with a median of 1.0%; for ID-LC-MS/MS, it was concentration-dependent, with a median of up to 8%. Weighted Deming regression gave mean slopes, intercepts, and correlation coefficients of 0.90-1.11, -0.055-0.013 nmol/L, and 0.993-0.997, respectively. The % difference plot showed between 7% and 26% of the results outside a total error limit of 14%, with median deviations from ID-GC-MS between -9.6 and 0.4%., Conclusions: This study demonstrated fairly good accuracy and standardization of the tested ID-LC-MS/MS procedures. Performance differences between procedures were evident in some instances, due to improper calibration and between-run calibration control. This emphasizes the need for thorough validation, including traceability, of new ID-LC-MS/MS procedures.

Background: An international working group convened by the American Diabetes Association (ADA) called for a reference measurement procedure for use in a trueness-based standardization project of insulin immunoassays. In view of this demand, we conducted a pilot study to investigate the feasibility of such a standardization project with our isotope dilution-liquid chromatography/tandem mass spectrometry (ID-LC/tandem MS) procedure., Methods: We evaluated the precision, accuracy, and limit of quantification (LoQ) of the ID-LC/tandem MS procedure by use of insulin-free serum supplemented with insulin to give 3 pools with concentrations of 0.0796, 0.769, and 5.56 microg/L. We conducted a pilot method comparison study with 4 immunoassays and 80 samples from fasting and glucose-stimulated patients., Results: The within-run and total imprecision (CV) ranged from 3.2% to 6.3% and from 4.9% to 12.1% (listing sequence from the high to the low pool). The recovery from supplemented insulin-free sera ranged from 101.8% to 104.1%, and the LoQ was 0.07 microg/L (12 pmol/L). Weighted Deming regression and correlation analysis of the method-comparison data showed considerable between-assay variation for the immunoassays but, with the exception of one assay, excellent correlation with ID-LC/tandem MS. Recalibration of the immunoassay results considerably reduced the between-assay variation. Moreover, after recalibration, 3 of the 4 assays fulfilled the total error specification of 32% proposed by the ADA Workgroup., Conclusions: Recalibration of insulin assays by regression equations established from method comparison with ID-LC/tandem MS can result in successful standardization and fulfillment of the total error criterion proposed by the ADA Workgroup.

Background: Serum-free thyroxine (FT4) testing is recommended for diagnosis or monitoring of thyroid dysfunction, particularly in cases of hormone binding abnormalities. However, the poor intermethod agreement among commercial FT4 assays suggests a need for standardization with a hierarchically higher measurement procedure. To that purpose, we applied equilibrium dialysis (ED) in combination with isotope dilution-liquid chromatography/tandem mass spectrometry (ID-LC-tandem MS)., Methods: After ED, we collected dialysate into tubes containing [13C6]-T4 for ID and [13C9]-T4 as carrier, purified the samples by solid-phase extraction, and analyzed them with LC/tandem MS. We evaluated the procedure's analytical performance and tested its suitability for measurement of hypo-, eu-, and hyperthyroid serum FT4 concentrations. We conducted a pilot method comparison study with 3 commercial assays to investigate whether frozen sera could be used for the purpose of FT4 standardization., Results: The within-run, between-run, and total CVs (inclusive ED) were 3.7%, 4.2%, and 5.6%, respectively (17.7 pmol/L; n = 20). The mean accuracy, estimated from recovery experiments with dialysate and dialysis buffer supplemented at 8.7, 18.7, and 33.5 pmol/L, and from analysis of certified sera gravimetrically diluted to 9.8, 19.2, and 34.8 pmol/L, was 98.0% to 102.8%. The procedure's limit of detection and limit of quantification were 0.5 and 1.3 pmol/L, respectively. The method comparison demonstrated the suitability of the selected sera for standardization of FT4 assays and confirmed the lack of assay comparability., Conclusions: We demonstrated that the described ED-ID-LC/tandem MS procedure and the selected type of sera qualify for standardization of FT4 measurements.

Background: Diagnostic manufacturers must ensure/document metrologically traceable assays. We report on a feasibility study of a split-sample comparison for that purpose. Processed, frozen single-donation sera, assigned target values by candidate reference measurement procedures (cRMPs), were used with immunoassays for total thyroxine (TT(4)) and triiodothyronine (TT(3)) as models., Methods: Two serum panels were quantified for TT(4) and TT(3) with validated cRMPs and measured in parallel with at least 14 immunoassays. The results were interpreted in terms of traceability of calibration (trueness) and of the individual measurement result (accuracy) by linear regression analysis and graphical representation against specifications. The commutability of the sera was investigated by parallel analysis of TT(4) in freshly collected but nonfiltered specimens., Results: The TT(4) (TT(3)) concentrations in the sera (according to the cRMPs) were 64-269 nmol/L (0.88-13.7 nmol/L). The method comparison showed that for TT(4), on average, the immunoassays produced results in agreement with the cRMPs, whereas for TT(3), results were typically higher. It also demonstrated a considerable between-assay divergence in traceability of calibration and accuracy. The evidence of noncommutability of the sera attributable to processing, however, indicates that the interpretation should be treated with caution., Conclusions: Frozen sera can be used for documenting/validating traceability of total thyroid measurements. The way in which the sera are processed may jeopardize commutability, however, and therefore requires in-depth investigation.

Background: Assuring/demonstrating metrologic traceability of in vitro diagnostics necessitates the availability of measurand-specific reference measurement systems (RMSs) and the possibility for industry to work with competent reference measurement laboratories (RMLs). Here we report the results of a European project to investigate the feasibility of developing a RMS for serum total thyroxine., Methods: Four candidate RMLs (cRMLs) developed/implemented variants of a candidate reference measurement procedure (cRMP) based on isotope dilution-liquid chromatography-mass spectrometry. The sole constraint implemented was calibration with a common thyroxine primary calibrator. The RMPs were externally validated and assessed for comparability in round-robin trials using common samples, i.e., 5 lyophilized and 33 frozen native sera. At the same time, the performance of the cRMLs organized in a network was assessed. For uniform external quality assessment, common performance specifications were agreed on., Results: All cRMLs performed the cRMPs with fulfillment of the predefined specifications: total and between-laboratory CVs < or =2.0% and 2.5%, respectively, and a systematic deviation < or =0.9%, estimated with a target assigned from the mean of means obtained by the cRMLs. The mean expanded uncertainty for value assignment to the native sera was 2.1%., Conclusions: A network of cRMLs, with externally conformed competence to properly perform RMPs, has been established. Performance specifications were defined and will form the basis for admittance of new network members. A serum panel, successfully targeted during the validation process, is available for split-sample measurements with commercial routine measurement procedures. The model can now be used for other measurands for which traceability to the Systeme International d'Unites is needed.

Background: To assess the analytical validity of free testosterone (FTe) measurements, a reference measurement procedure (RMP) is required. For steroids, isotope dilution-mass spectrometry is accepted as state-of-the-art technology. Because FTe is defined as the hormone fraction in serum water in equilibrium with the protein-bound fraction, the RMP should include a physical separation step. The use of equilibrium dialysis (ED) or ultrafiltration (UF) is advocated. Our objective was to develop such a candidate RMP., Methods: We selected UF combined with isotope dilution-gas chromatography-mass spectrometry (ID-GC/MS) for direct measurement of Te in the ultrafiltrate. After optimization of the UF process, the complete procedure was validated by use of split-sample comparisons with indirect ED (iED) and symmetric dialysis (SyD)., Results: The candidate RMP gave maximum within-day, between-day, and total CVs of 3.0%, 3.1%, and 4.3%. The Deming regression equations for the respective method comparisons were: UF-ID-GC/MS = 0.98(iED) - 53 pmol/L (r = 0.94; S(y|x)= 42 pmol/L) and UF-ID-GC/MS = 0.92(SyD) + 21 pmol/L (r = 0.97; S(y|x)= 31 pmol/L)., Conclusions: We achieved the objective of a state-of-the-art candidate RMP, which agreed well with iED and SyD. However, we also demonstrated that a degree of discordance remains, which may require a decision from an authoritative organization on the recommended procedure to measure free hormone concentrations.

This manuscript explains the establishment and validation of metrological traceability of calibration for routine measurement procedures using common medical decision-making criteria. Metrological traceability is considered the basis for achieving comparability of measurement results in laboratory medicine. This concept is supported by European legislation, which demands that manufacturers provide assurance and demonstrate metrological traceability of in vitro Diagnostic Medical Devices. The guidance to comply with these legislative requirements is available in different CEN/ISO standards and is used as a basis of this manuscript. The goals and accomplishments in metrological traceability of SI- and non-SI analytes is considered. Specific problems, such as non-availability of primary reference materials and measurement procedures, lack of official endorsement, and non-commutability of certain reference materials are discussed. With respect to non-commutability, the use of split-sample measurements is advocated. Also, the expression of measurement uncertainty associated with the application of the metrological traceability chain is discussed. In addition, the need for post-market vigilance assessment of traceable performance is considered. Finally, laboratory medicine scientific and professional societies, diagnostics manufacturers, and clinicians are urged to share responsibilities for understanding the implications of metrological traceability of routine measurements.

Background: In clinical chemistry, traceability of measurements is of high priority., Methods: In this literature review, current recommendations on the process of establishing traceability (or standardization) are critically discussed., Results: Traceability is to be established to the highest international standards by a comprehensive reference measurement system. Elementary to this system are a metrological basis, a measurement unit system, i.e., the Système International d'Unités (SI), its embodiment by a material standard and a calibration hierarchy for transfer of accuracy/trueness to the manufacturer's product calibrators and routine methods. However, for analytes lacking an unequivocally recognized entity, the International Unit (IU) and International Standard (IS) concept have been developed. On this basis, the review distinguishes between traceability of SI- and IU-analytes., Conclusions: SI-traceability, exemplified by cortisol, is straightforward. However, special attention is needed for "free analytes" and "analyte families". For traceability of IU-analytes, exemplified by hCG, the standardization process passes different phases in function of the history of the analyte (discovery, ISs, measurement by bioassays/immunoassays, complete structure elucidation). However, perspectives to the development of an SI-based reference measurement system are realistic. Last but not least, for successful global implementation of the standardization process, consensus of all major players in the field will be required.