Search

Correct interpretation of thyroid function tests relies on correct reference intervals (RIs) for thyroid-stimulating hormone (TSH) and free thyroxine (FT4). ISO15189 mandates periodic verification of RIs, but laboratories struggle with cost-effective approaches. We investigated whether indirect methods (utilizing historical laboratory data) could replace the direct approach (utilizing healthy reference individuals) and compared results with manufacturer-provided RIs for TSH and FT4. We collected historical data (2008–2022) from 13 Dutch laboratories to re-establish RIs by employing indirect methods, TMC (for TSH) and refineR (for FT4). Laboratories used common automated platforms (Roche, Abbott, Beckman or Siemens). Indirect RIs (IRIs) were determined per laboratory per year and clustered per manufacturer (>1.000.000 data points per manufacturer). Direct RIs (DRIs) were established in 125 healthy individuals per platform. TSH IRIs remained robust over the years for all manufacturers. FT4 IRIs proved robust for three manufacturers (Roche, Beckman and Siemens), but the IRI upper reference limit (URL) of Abbott showed a decrease of 2 pmol/L from 2015. Comparison of the IRIs and DRIs for TSH and FT4 showed close agreement using adequate age-stratification. Manufacturer-provided RIs, notably Abbott, Roche and Beckman exhibited inappropriate URLs (overall difference of 0.5–1.0 µIU/mL) for TSH. For FT4, the URLs provided by Roche, Abbott and Siemens were overestimated by 1.5–3.5 pmol/L. These results underscore the importance of RI verification as manufacturer-provided RIs are often incorrect and RIs may not be robust. Indirect methods offer cost-effective alternatives for laboratory-specific or platform-specific verification of RIs. [ABSTRACT FROM AUTHOR]

With the upcoming EU regulation on the use of in-vitro diagnostic devices, a critical evaluation of the current status of our in-house developed LC-MS/MS methods is timely and of great relevance. Recently, much attention has been devoted to the need for better specification of analytical and clinical performance. Appropriate reporting of the actual achieved analytical performance is an important determinant of the clinical performance and subsequent clinical effectiveness of a test. We advocate for the application of CLSI C62-A guidelines for method validation and suggest some adaptations for analytical validation of in-house developed LC-MS/MS methods for endogenous substances. Additionally, we underline the importance of well-equipped reviewers and standardized method description, including the presentation of figural evidence of obtained method performance. Achieving this ensures future quality of our in-house developed LC-MS/MS methods. [ABSTRACT FROM AUTHOR]

Background The variety of LC-MS/MS methods measuring total 25(OH)D used today is vast and the comparability among these methods is still not well assessed. Methods Here, we performed a comparison in samples of healthy donors between the currently routinely used 25(OH)D LC-MS/MS methods in the Netherlands and the Ghent University reference measurement procedure to address this issue (n = 40). Additionally, an interlaboratory comparison in patient serum samples assessed agreement between the Dutch diagnostic methods (n = 37). Results The overall correlation of the routine methods for 25(OH)D 3 with the reference measurement procedures and with the mean of all diagnostic methods was excellent (r > 0.993 and r > 0.989, respectively). Three out of five methods aligned perfectly with both the reference measurement procedure and the median of all methods. One of the routine methods showed a small positive bias, while another showed a small negative bias consistently in both comparisons. Conclusion The biases most probably originated from differences in calibration procedure and may be obviated by reassessing calibration of stock standards and/or calibrator matrices. In conclusion, five diagnostic centers have performed a comparison with the 25(OH)D Ghent University reference measurement procedure in healthy donor serum samples and a comparison among themselves in patient serum samples. Both analyses showed a high correlation and specificity of the routine LC-MS/MS methods, yet did reveal some small standardization issues that could not be traced back to the technical details of the different methods. Hence, this study indicates various calibration procedures can result in perfect alignment. [ABSTRACT FROM AUTHOR]

Background To assess a patient’s vitamin D status the precursor metabolite 25-hydroxyvitamin D can be determined. However, measurement of 1,25-dihydroxyvitamin D is required when disorders of 1a-hydroxylation, extrarenal 1a-hydroxylation, or vitamin D receptor defects are suspected. Methods The aim of this study was to determine reference values for 1,25-dihydroxyvitamin D 3 and D 2 using a 2D ID-UPLC–MS/MS method. Results The LC–MS/MS method, able to measure picomolar concentrations of both 1,25-dihydroxyvitamin D 3 and D 2 in human serum, was extensively validated. Intra-assay variations were <5% and 8.5% and <7.5% and 11%, for 1,25-dihydroxyvitamin D 3 and D 2 , respectively, over the whole dynamic range (3.1–376 and 3.1–652 pmol/L). Limit of quantitation was 3.4 pmol/L for both compounds. Our method correlated well with a published LC–MS/MS method ( r = 0.87) and with the average 1,25-dihydroxyvitamin D 3 results of the vitamin D External Quality Assessment Scheme (DEQAS) determined with LC–MS/MS ( r = 0.93). Reference ranges, determined in 96 plasma samples of healthy volunteers were 59–159 pmol/L and <17 pmol/L for respectively 1,25-dihydroxyvitamin D 3 and D 2 . The female part of the reference group showed a statistically significant decrease of 1,25-dihydroxyvitamin D 3 concentrations with age. The presence of significantly higher average 1,25-dihydroxyvitamin D 3 levels in premenopausal women taking oral contraceptive pills compared to postmenopausal women suggests that this effect is estrogen-related, as estrogens lead to a higher vitamin D binding protein. Conclusions The major finding of the present study is a reference interval of 59–159 pmol/L for 1,25-dihydroxyvitamin D 3 determined with a highly sensitive and precise LC–MS/MS method. [ABSTRACT FROM AUTHOR]

Keywords: 24,25-dihydroxyvitamin D3; 25-hydroxyvitamin D; infantile idiopathic hypercalcemia; LC-MS/MS; reference values The ratio 25(OH)D/24,25(OH) SB 2 sb D was calculated using 25(OH)D SB 3 sb and 24,25(OH) SB 2 sb D SB 3 sb . To the best of our knowledge, this is the first time reference values for 24,25(OH) SB 2 sb D SB 3 sb and the 25(OH)D/24,25(OH) SB 2 sb D ratio have been established with a method standardized using the NIST SRM 972a reference material. In conclusion, using a newly developed and standardized LC-MS/MS assay, we have established reference values for the 25(OH)D/24,25(OH) SB 2 sb D ratio in both men and women, aged 20-71 years. [Extracted from the article]

Several publications have reported on the pre-analytical stability of fibroblast growth factor 23 (FGF23) and some recommend coating blood collecting tubes with protease inhibitors, in order to prevent degradation. These recommendations are based on observations for a first generation assay for the measurement of intact FGF23. However, if this also applies for the contemporary immunoassays, and at what stage of pre-analysis, is unknown. We reviewed data from these previous reports on the issue of FGF23 stability and complemented these findings with data from novel experiments. We concluded that the contemporary intact FGF23 assays by Immutopics, Kainos, Millipore and DiaSorin do not suffer from immediate loss of FGF23 signal and do not require blood withdrawal in protease inhibitor-coated collecting tubes. Nevertheless, FGF23 concentrations do decline when centrifugation is delayed up to 8 h and prompt centrifugation is therefore advised. • None of the contemporary intact FGF23 immunoassays suffer from immediate loss of FGF23 signal. • With the contemporary immunoassays there is no need to coat blood collecting tubes with protease inhibitors. • Prompt centrifugation is advised as signal declines if centrifugation is delayed. [ABSTRACT FROM AUTHOR]

25-hydroxyvitamin D (25(OH)D) is commonly measured to assess vitamin D status. Other vitamin D metabolites such as 24,25-dihydroxyvitamin D (24,25(OH)2D) provide additional insights into vitamin D status or metabolism. Earlier studies suggested that the vitamin D metabolite ratio (VMR), calculated as 24,25(OH)2D/25(OH)D, could predict the 25(OH)D increase after vitamin D supplementation. However, the evidence for this additional value is inconclusive. Therefore, our aim was to assess whether the increase in 25(OH)D after supplementation was predicted by the VMR better than baseline 25(OH)D. Plasma samples of 106 individuals (25(OH)D < 75 nmol/L) with hypertension who completed the Styrian Vitamin D Hypertension Trial (NC.T.02136771) were analyzed. Participants received vitamin D (2800 IU daily) or placebo for 8 weeks. The treatment effect (ANCOVA) for 25(OH)D3, 24,25(OH)2D3 and the VMR was 32 nmol/L, 3.3 nmol/L and 0.015 (all p < 0.001), respectively. Baseline 25(OH)D3 and 24,25(OH)2D3 predicted the change in 25(OH)D3 with comparable strength and magnitude. Correlation and regression analysis showed that the VMR did not predict the change in 25(OH)D3. Therefore, our data do not support routine measurement of 24,25(OH)2D3 in order to individually optimize the dosage of vitamin D supplementation. Our data also suggest that activity of 24-hydroxylase increases after vitamin D supplementation. [ABSTRACT FROM AUTHOR]

We now have the ability to measure a number of different vitamin D metabolites with very accurate methods. The most abundant vitamin D metabolite, 25-hydroxyvitamin D, is currently the best marker for overall vitamin D status and is therefore most commonly measured in clinical medicine. The added value of measuring metabolites beyond 25-hydroxyvitamin D, like 1,25-, and 24,25-dihydroxyvitamin D is not broadly appreciated. Yet, in some more complicated cases, these metabolites may provide just the information needed for a legitimate diagnosis. The problem at present, is knowing when to measure, what to measure and how to measure. For 25-hydroxyvitamin D, the most frequently used automated immunoassays do not meet the requirements of today’s standards for certain patient groups and liquid chromatography-tandem mass spectrometry is the desired method of choice in these individuals. The less frequently measured 1,25-dihydroxyvitamin D metabolite enables us to identify a number of conditions, including 1α-hydroxylase deficiency, hereditary vitamin D-resistant rickets and a number of granulomatous diseases or lymphoproliferative diseases accompanied by hypercalcaemia. Furthermore, it discriminates between the FGF23-mediated and non-FGF23-mediated hypophosphatemic syndromes. The 24,25-dihydroxyvitamin D metabolite has proven its value in the diagnosis of idiopathic infantile hypercalcaemia and has the potential of having value in identifying other diseases. For both metabolites, the understanding of the origin of differences between assays is limited and requires further attention. Nonetheless, in every way, appropriate measurement of vitamin D metabolism in the clinical laboratory hinges eminently on the comprehension of the value of the different metabolites, and the importance of the choice of method. [ABSTRACT FROM AUTHOR]

Background: Thyroid-stimulating hormone (TSH) and subsequent free thyroxine (FT4) concentrations outside the reference interval (RI) are used to diagnose thyroid diseases. Most laboratories do not provide age-specific RIs for TSH and FT4 beyond childhood, although TSH concentrations vary with age. Therefore, we aimed to establish TSH and FT4 age-specific RIs throughout life and aimed to determine whether using these RIs would result in reclassification of thyroid disease diagnoses in adults. Methods: This multicenter retrospective cross-sectional study used big data to determine indirect RIs for TSH and FT4. These RIs were determined by TMC and refineR-analysis, respectively, using four different immunoassay platforms (Roche, Abbott, Siemens, and Beckman Coulter). Retrospective data (2008-2022) from 13 Dutch laboratories for general practitioners and local hospitals were used. RIs were evaluated per manufacturer. Age groups were established from 2 to 20 years by 2-year categories and decade categories between 20 and 100 years. Results: We included totally 7.6 million TSH and 2.2 million FT4 requests. TSH upper reference limits (URLs) and FT4 lower reference limits were higher in early childhood and decreased toward adulthood. In adulthood, TSH URLs increased from 60 years in men, and from 50 years in women, while FT4 URLs increased from 70 years onward. Using adult age-specific RIs resulted in a decrease in diagnoses of subclinical and overt hypothyroidism in women above 50 and men above 60 years in our Roche dataset. Conclusion: This study stressed the known importance of using age-specific RIs for TSH and FT4 in children. This study also showed the clinical relevance of using age-specific RIs for TSH in adulthood to reduce diagnoses of subclinical hypothyroidism in older persons. Therefore, implementation of adult TSH age-specific RIs should be strongly considered. Data are less uniform regarding FT4 age-specific RIs and more research should be performed before implementing these in clinical practice.

The measurement of vitamin D metabolites aids in assessing vitamin D status and in diagnosing disorders of calcium homeostasis. Most laboratories measure total 25-hydroxyvitamin D (25(OH)D), while others have taken the extra effort to measure 25(OH)D2 and 25(OH)D3 separately and additional metabolites such as 1,25-dihydroxyvitamin D and 24,25-dihydroxyvitamin D. The aim of this review is to provide an updated overview of the main markers of vitamin D metabolism, define the intended measurands, and discuss the advantages and disadvantages of the two most widely used assays, automated assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Whether using the easy and fast automated assays or the more complex LC-MS/MS, one should know the pitfalls of the used technique in order to interpret the measurements. In conclusion, automated assays are unable to accurately measure 25(OH)D in all patient groups, including persons using D2. In these cases, an LC-MS/MS method, when appropriately developed and standardized, produces a more reliable measurement.