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Objective: Serum prolactin measurements are important in the differential diagnosis of pituitary masses and subfertility. We observed discrepancies in serum prolactin levels in several patients measured with different immunoassays. Despite differences in assay results, the reference intervals (RIs) derived by the manufacturers were similar. In this study, we aimed to investigate prolactin assay differences and to re-establish RIs for different prolactin immunoassays., Methods: For the assay comparison, serum samples were collected from men and women visiting the Amsterdam UMC hospital. Prolactin levels were measured using the AtellicaTM IM analyzer (Siemens Healthineers) and the Cobas (Roche Diagnostics) immunoassay. RIs for prolactin were re-established for men, premenopausal women, and postmenopausal women for both the Atellica and Cobas immunoassay., Results: Prolactin levels measured using the Cobas immunoassay were 1.75 times higher than those measured using the Atellica immunoassay. The re-established RIs for Atellica and Cobas confirmed these findings and were <0.32 U/L; <0.55 U/L for men; <0.64 U/L; <0.86 U/L for premenopausal women, and <0.31 U/L; <0.59 U/L for postmenopausal women, respectively, for Atellica and Cobas assays. The re-established RIs of the Atellica assay matched the current and manufacturer RIs, whereas those for Cobas differed substantially., Conclusions: Prolactin levels are assay-dependent, and the re-established RIs are different for the Atellica and Cobas assays. We recommend that laboratory specialists and manufacturers periodically review prolactin assay RIs, as incorrect RIs can lead to misinterpretation of prolactin levels and unnecessary referrals and further laboratory testing, as we have experienced., Plain Language Summary: We showed that the results of different prolactin tests disagree by 75%, which hinders correct interpretation. Thus, we established test-specific prolactin normal values. By being aware of test differences and using test-specific normal values, one can ensure correct interpretation and prevent unnecessary referrals and concern.

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 aim of this study was to investigate the associations among sex hormone-binding globulin (SHBG), visceral adipose tissue (VAT), liver fat content, and risk of type 2 diabetes (T2D). In the Netherlands Epidemiology of Obesity study, 5,690 women (53%) and men (47%) without preexisting diabetes were included and followed for incident T2D. SHBG concentrations were measured in all participants, VAT was measured using MRI, and liver fat content was measured using proton magnetic resonance spectroscopy in a random subset of 1,822 participants. We examined associations between SHBG and liver fat using linear regression and bidirectional Mendelian randomization analyses and between SHBG and T2D using Cox regression adjusted for confounding and additionally for VAT and liver fat to examine mediation. Mean age was 56 (SD 6) years, mean BMI was 30 (SD 4) kg/m2, median SHBG was 47 (interquartile range [IQR] 34-65) nmol/L in women and 34 (26-43) nmol/L in men, and median liver fat was 3.4% (IQR 1.6-8.2%) in women and 6.0% (2.9-13.5%) in men. Compared with the highest SHBG quartile, liver fat was 2.9-fold (95% CI 2.4, 3.4) increased in women and 1.6-fold (95% CI 1.3, 1.8) increased in men, and the hazard ratio of T2D was 4.9 (95% CI 2.4, 9.9) in women and 1.8 (1.1, 2.9) in men. Genetically predicted SHBG was associated with liver fat content (women: SD -0.45 [95% CI -0.55, -0.35]; men: natural logarithm, -0.25 [95% CI -0.34, -0.16]). VAT and liver fat together mediated 43% (women) and 60% (men) of the SHBG-T2D association. To conclude, in a middle-aged population with overweight, the association between low SHBG and increased risk of T2D was, for a large part, mediated by increased VAT and liver fat., (© 2024 by the American Diabetes Association.)

Objectives: Congenital adrenal hyperplasia (CAH) is a rare, inherited disorder of adrenal steroid synthesis. In many countries it is part of the neonatal screening program enabling early diagnosis and treatment. In case of an abnormal neonatal screening result or when other differences of sexual development (DSD) are suspected, measurement of serum steroid hormones using liquid chromatography coupled to mass spectrometry (LC-MS/MS) is needed for further diagnosis. However, reliable age- and sex-specific reference intervals (RIs) for serum steroid hormones during the neonatal period are missing. We therefore aimed to establish LC-MS/MS based RIs for serum steroid hormones in neonates., Methods: Serum was obtained from healthy term neonates at two time points: 130 samples at day 3-8 ( T1 , time of the neonatal screening) and 126 samples at day 13-15 ( T2,  two weeks old). Concentrations of cortisol, cortisone, corticosterone, 11-deoxycortisol, 21-deoxycortisol, 11-deoxycorticosterone, testosterone, androstenedione, and 17-hydroxyprogesterone (17-OHP) were measured using LC-MS/MS., Results: RIs (in nmol/L) were established for T1 and T2 : cortisone (19.3-215;18.0-212), cortisol (10.0-407;8.4-446), corticosterone (<31;<50), 11-deoxycortisol (0.73-4.6;0.70-3.6), 17-OHP (<4.9;<5.1), androstenedione (0.3-1.8;0.3-2.7), 11-deoxycorticosterone (<0.2;<0.2), and 21-deoxycortisol (<1;<1), respectively. Testosterone differed between boys and girls: RIs at T1 and T2 for boys were 0.27-4.3 and 0.63-13.9, and for girls<0.30 and <0.47, respectively., Conclusions: We established LC-MS/MS based RIs for cortisol, cortisone, corticosterone, 11-deoxycortisol, 21-deoxycortisol, 11-deoxycorticosterone, testosterone, androstenedione, and 17-OHP in neonates in the first and second week of life., (© 2024 the author(s), published by De Gruyter, Berlin/Boston.)

Inconclusive and false-positive newborn screening (NBS) results can cause parental stress and increase healthcare expenditures. These results can be reduced by improving NBS algorithms. This was recently done for Congenital Hypothyroidism (CH), Congenital Adrenal Hyperplasia (CAH) and Maple Syrup Urine Disease (MSUD) in the Dutch NBS program. The current study estimates the financial consequences of these improved algorithms related to the reduction in inconclusive results and false-positives. For each improved algorithm, the care pathway of an inconclusive/false-positive result was analyzed. The costs associated with the improvements, based on the change in inconclusive results/false-positives, were assessed to estimate the cost reduction per year. The improvements resulted in a reduction of inconclusive results and/or false-positives, without increasing false-negatives. For CH, false positives decreased by 26 per year with a related cost reduction of EUR 31,156. For CAH, 95 second heel punctures and seven false-positives per year were avoided, leading to a related cost reduction of EUR 7340. For MSUD, five false-positives per year were avoided with a related cost reduction of EUR 11,336. The improved screening algorithms led to a cost reduction of EUR 49,832 annually. Together with the known negative psychosocial effects associated with an inconclusive or false-positive NBS result, these results highlight the importance of improving NBS algorithms.

Thyroid cancer is the most prevalent endocrine cancer. Prognosis depends on type and stage of cancer when discovered. Treatment involves (hemi-)thyroidectomy, possibly followed by additional therapeutic options. After treatment, patients are monitored using serum concentrations of endocrine tumour marker thyroglobulin in case of differentiated cancer and calcitonin in case of medullary thyroid cancer. Measuring thyroglobulin and calcitonin concentrations in serum may be challenging. In this review we give a complete overview of the evolvement in laboratory measurements regarding thyroglobulin and calcitonin with an emphasis on (pre-)analytical challenges and potential approaches to overcome current pitfalls.

Rationale & Objective: Kidneys are vital for vitamin D metabolism, and disruptions in both production and catabolism occur in chronic kidney disease. Although vitamin D activation occurs in numerous tissues, the kidneys are the most relevant source of circulating active vitamin D. This study investigates extrarenal vitamin D activation and the impact of kidney transplantation on vitamin D metabolism in patients who are anephric., Study Design: Case series., Setting & Participants: Adult patients with previous bilateral nephrectomy (anephric) not receiving active vitamin D therapy evaluated at the time of (N=38) and 1 year after (n=25) kidney transplantation., Analytical Approach: Chromatography with tandem mass spectrometry was used to measure vitamin D metabolites. Activity of CYP24A1 [24,25(OH) 2 D/25(OH)D] and CYP27B1 [1α,25(OH) 2 D/25(OH)D] is expressed as metabolic ratios. Differences between time points were evaluated by paired t-test or Wilcoxon matched-pairs signed-rank test., Results: At time of transplantation, 1α,25(OH) 2 D was detectable in all patients (4-36pg/mL). There was a linear relationship between 25(OH)D and 1α,25(OH) 2 D levels (r=0.58, P<0.001), with 25(OH)D explaining 34% of the variation in 1α,25(OH) 2 D levels. There were no associations between 1α,25(OH) 2 D and biointact parathyroid hormone (PTH) or fibroblast growth factor 23 (FGF-23). One year after transplantation, 1α,25(OH) 2 D levels recovered (+205%), and CYP27B1 activity increased (+352%). Measures of vitamin D catabolism, 24,25(OH) 2 D and CYP24A1 activity increased 3- to 5-fold. Also, at 12 months after transplantation, 1α,25(OH) 2 D was positively correlated with PTH (ρ=0.603, P=0.04) but not with levels of 25(OH)D or FGF-23., Limitations: Retrospective, observational study design with a small cohort size., Conclusions: Low-normal levels of 1α,25(OH) 2 D was demonstrated in anephric patients, indicating production outside the kidneys. This extrarenal CYP27B1 activity may be more substrate driven than hormonally regulated. Kidney transplantation seems to restore kidney CYP27B1 and CYP24A1 activity, as evaluated by vitamin D metabolic ratios, resulting in both increased vitamin D production and catabolism. These findings may have implications for vitamin D supplementation strategies in the setting of kidney failure and transplantation., Plain-Language Summary: Vitamin D activation occurs in multiple tissues, but the kidneys are considered the only relevant source of circulating levels. This study investigates vitamin D activation outside the kidneys by measuring vitamin D metabolites in 38 patients without kidneys. Active vitamin D was detectable in all patients, indicating production outside of the kidneys. There was a strong relationship between active and precursor vitamin D levels, but no association with mineral metabolism hormones, indicating that vitamin D production was more substrate dependent than hormonally regulated. One year after kidney transplantation, active vitamin D levels increased 2-fold and breakdown products increased 3-fold, indicating that production and degradation of the hormone recovers after kidney transplantation. These findings are relevant for future research into vitamin D supplementation in kidney failure., (Copyright © 2024 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.)

Purpose: Graves' disease (GD) is an auto-immune cause of hyperthyroidism. First-line treatment often consists of a 12-18 month course of antithyroid drugs (ATD). After discontinuation of ATD, GD relapses in approximately 50% of patients. The 'Graves recurrent event after therapy+ ' (GREAT+) score may predict individual relapse chances after ATD discontinuation more accurately based on clinical and laboratory parameters at diagnosis. We investigated the need for the GREAT+ score through an online questionnaire among GD patients and physicians treating GD., Methods: An anonymous online questionnaire was distributed to patients and physicians between June 2022 and August 2023., Results: The questionnaire was completed by 532 patients and 44 physicians. Results showed that 94% of patients were interested in knowing their GREAT+ score at the start of treatment. 55% would consider definite treatment (radioiodine/thyroidectomy) as first-line treatment in case of a high relapse chance. 98% of the physicians indicated the GREAT + score would support patient counseling. 84% may change their advice for first-line treatment if a patient has a high relapse chance based on the score., Conclusion: Patients and physicians considered the GREAT+ score as a valuable addition to the current available information which could change treatment decisions. Therefore, external validation of the GREAT+ score is justified to implement this score in clinical practice., (© 2024. The Author(s).)

Background: Vitamin D, acknowledged since the 1930s for its role in preventing rickets, gained additional prominence in relation to fragility fracture prevention in the late 1980s. From the early 2000s, connections between vitamin D deficiency and extra-skeletal pathologies emerged, alongside increased awareness of widespread deficits. This prompted crucial debates on optimal serum concentrations, expected to conclude when the outcomes of high-dose supplementation randomized controlled trials were available. Skepticism arose with inconclusive results from these trials., Content: This review begins with an exploration of vitamin D metabolism, followed by a detailed description of the measurement of vitamin D metabolites and the crucial role of standardization. Subsequent sections focus on the association of vitamin D with bone health and explore the extra-skeletal effects. The review concludes with a comprehensive discussion on the definition of vitamin D status and its implications for supplementation., Summary: Despite standardization efforts, assay variations and challenges still exist, especially in specific patient groups. Vitamin D supplementation has a significant impact on bone metabolism and optimal vitamin D status improves the efficacy of antiresorptive drugs such as bisphosphonates. The extra-skeletal effects of vitamin D remain debated, but may include potential benefits in conditions such as respiratory infections and cancer mortality, particularly in deficient individuals. The definition of vitamin D sufficiency is nuanced, especially when variations in population groups and analytical methods are taken into account. Despite ongoing debates and recent mega-trials tempering enthusiasm, vitamin D remains a complex and essential element in human health. Further research is needed to clarify its role in various health outcomes and guide supplementation strategies., (© Association for Diagnostics & Laboratory Medicine 2024. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Leptin and ghrelin have been linked to depressive symptoms in older adults. There is a large overlap between depression and anxiety in this group. It is unclear whether the same associations exist with anxiety. Adiponectin has an inverse association with anxiety in older adults. However, the association between the most biologically active isoform - high-molecular-weight (HMW) adiponectin - and anxiety has not been previously reported., Methods: We analyzed the association between leptin, ghrelin and HMW adiponectin and general symptoms of anxiety (HADS-A score ≥ 7) at baseline and after three years of follow-up in a population based cohort of older adults in the Netherlands (n = 898) using multivariable logistic regression analyses., Results: For leptin there was significant effect modification by sex. We found a positive association between leptin and general symptoms of anxiety in men at baseline and after three years of follow-up after adjusting for depressive symptoms, when comparing the third to the first leptin tertile (T3 vs T1 OR 3.40, 95 % CI 1.08 - 10.78). We found no significant associations for ghrelin. HMW adiponectin was associated with general symptoms of anxiety at follow up. We found a positive association both before and after adjustment for depressive symptoms (T3 vs T1 OR 3.26, 95 % CI 1.36 - 7.83)., Conclusions: Our results showed significant associations in men only between leptin and HMW adiponectin and general symptoms of anxiety after three years of follow up. Our findings contribute to further insight into the pathophysiology of anxiety in older adults. However, further research is necessary as we show associations., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Longitudinal Aging Study Amsterdam reports financial support was provided by Netherlands Ministry of Health Welfare and Sport. Prof. dr. M.L. Drent reports financial support was provided by Ipsen Pharmaceuticals Netherlands. The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objective: Transgender women who underwent gonadectomy have lower serum testosterone concentrations than cisgender women. There is uncertainty regarding the dosing and side effects of supplementation of testosterone in transgender women. This study aimed to assess the feasibility of dosing testosterone to the cisgender female physiological range in transgender women. In addition, we explored changes in cardiovascular parameters, virilizing side effects, and clinical symptoms., Design: This is an open-label, single-arm feasibility study. Participants initially went through a dose-titration phase with 2-week intervals of 0.07-0.09-0.13 mL (277-318-403 μg bioavailable testosterone) testosterone 2% gel to establish a dose leading to serum testosterone concentrations between 1.5 and 2.5 nmol/L. This dose was then continued for 8 weeks., Methods: Participants applied daily transdermal testosterone 2% gel (Tostran®) at the prescribed dosage. Testosterone was measured every 2-4 weeks. Laboratory analyses, side effects, and clinical symptoms were evaluated., Results: In total, 12 participants were included. Most participants required a dose of 0.07 mL (277 μg bioavailable testosterone) or 0.09 mL (318 μg bioavailable testosterone) to reach serum testosterone concentrations of 1.5-2.5 nmol/L. Continuing this dose, testosterone concentrations remained stable throughout the study. Changes in clinical outcomes were in the desired direction, and side effects were mild., Conclusions: The use of testosterone supplementation in transgender women seems feasible and safe in the short term. Although dosing requires personalized titration, stable testosterone levels can be established. A blinded, placebo-controlled, randomized clinical trial is needed to study the clinical benefit., Competing Interests: Conflict of interest: None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Endocrinology.)

Background and Aims: Implementation of screening modalities have reduced the burden of colorectal cancer (CRC), but high false positive rates pose a major problem for colonoscopy capacity. We aimed to create a tailored screening algorithm that expands the fecal immunochemical test (FIT) with a blood specimen and current age to improve selection of individuals for diagnostic colonoscopy., Methods: In this prospective multicenter study, 8 blood-based biomarkers (carcinoembryonic antigen, ferritin, high-sensitivity C-reactive protein, human epididymis protein 4, Cyfra21-1, hepsin, interleukin 8, and osteoprotegerin) were investigated in 1977 FIT-positive individuals from the Danish national CRC screening program undergoing follow-up colonoscopy. Specimens were analyzed on Architect i2000, Architect c8000 (both from Abbott, Chicago, IL, USA), or Luminex xMAP machines (MilliporeSigma, St. Louis, Mo, USA). FIT analyses and blood-based biomarker data were combined with clinical data (ie, age and colonoscopy findings) in a cross-validated logistic regression model (algorithm) benchmarked against a model solely using the FIT result (FIT model) applying different cutoffs for FIT positivity., Results: The cohort included individuals with CRC (n = 240), adenomas (n = 938), or no neoplastic lesions (n = 799). The cross-validated algorithm combining the 8 biomarkers, quantitative FIT result, and age performed superior to the FIT model in discriminating CRC versus non-CRC individuals (area under the receiver operating characteristic curve, 0.77 vs 0.67, respectively; P < .001). When discriminating individuals with either CRC or high- or medium-risk adenomas versus low-risk adenomas or clean colorectum, the areas under the receiver operating characteristic curve were 0.68 versus 0.64 for the algorithm and FIT model, respectively., Conclusions: The algorithm presented here can improve patient allocation to colonoscopy, reducing colonoscopy burden without compromising cancer and adenoma detection rates or vice versa., Competing Interests: Disclosure The following authors disclosed financial relationships: C. Therkildsen: Research grants from Abbott Diagnostics Division and New Day Diagnostics. J. Liggett: Full-time employee of New Day Diagnostics. C. M. T. Beertsen: Research grant from Abbott Diagnostics Division. S. Gawel: Full-time employee of Abbott Diagnostics Division. E. Mayer: Full-time employee of New Day Diagnostics. G. J. Davis: Full-time employee of Abbott Diagnostics Division. All analyses were made blinded to J. Liggett, S. Gawel, E. Mayer, and G. J. Davis, whereafter molecular data were merged with clinical data by the academic team at Copenhagen University Hospital, Hvidovre, which also performed statistical analyses. Although statistical analyses were discussed with the industrial partners, the academic research team at Hvidovre Hospital decided on and performed the formal analyses independently. All other authors disclosed no financial relationships. Patient inclusion, sample collection and storage, and salary for all of the clinical and academic research team was provided by external funding granted to Hans Jørgen Nielsen and the Colorectal Cancer Research Unit at Hvidovre Hospital. This work was supported by the Andersen Foundation, Augustinus Foundation, Beckett Foundation, Inger Bonnéns Fund, Hans and Nora Buchards Fund, Walter Christensen Fund, P. M. Christiansen Fund, Kong Chr. X’s Fund, Aase and Ejnar Danielsens Fund, Family Erichsens Fund, Knud and Edith Eriksens Fund, Svend Espersens Fund, Elna and Jørgen Fagerholts Cancer Research Foundation, Sofus Carl Emil Friis Scholarship, Torben and Alice Frimodts Fund, Eva and Henry Frænkels Fund, Gangsted Foundation, Thora and Viggo Groves Memorial Scholarship, H-Foundation, Erna Hamiltons Scholarship, Søren and Helene Hempels Scholarship, Sven and Ina Hansens Fund, Henrik Henriksen Fund, Carl and Ellen Hertz Scholarship, Jørgen Holm and Elisa F. Hansen Memorial Scholarship, Jochum Foundation, Kurt and Ingeborg Daell's Foundation, Kornerup Foundation, Linex Foundation, Dagmar Marshalls Fund, Midtjyske Fund, Axel Muusfeldts Fund, Børge Nielsens Fund, Michael Hermann Nielsens Memorial Scholarship, Arvid Nilssons Fund, Obelske Family Fund, Krista and Viggo Petersens Fund, Willy and Ingeborg Reinhards Fund, Kathrine and Vigo Skovgaards Fund, Toyota Foundation, Vissing Foundation, P. Carl Petersen Fund, Danish Cancer Research Foundation, and Hvidovre Hospitals Research Fund. Furthermore, the collaborative companies, Abbott Diagnostics Division and New Day Diagnostics, have sponsored the study by providing the reagents, facilities, and staff required for the molecular analyses as well as a sample fee to Hvidovre Hospital for sample requisition., (Copyright © 2024 American Society for Gastrointestinal Endoscopy. All rights reserved.)

Objectives: Steroid hormone levels of patients may be monitored via dried blood spot (DBS) sampling at home. Stability of steroid hormones in DBS samples, however, needs to be established., Methods: DBS samples from healthy volunteers were collected and stored at various temperatures. Steroid hormone concentrations in DBS were measured directly, at day 2, day 7 and day 14 following storage at 37 °C and after 7 days, 14 days, 3 months and 6 months following storage at -20 °C, 4 °C and room temperature (RT). Cortisol, cortisone, corticosterone, testosterone, androstenedione, and 17-hydroxyprogesterone (17-OHP) were assessed using LC-MS/MS., Results: All steroids were stable (±15 %) up to 14 days when stored at 37 °C, except for cortisone (only stable until 2 days). All steroids were stable up to 6 months when stored at -20 °C, 4 °C and RT. However, there were some exceptions, for androstenedione at RT (only stable until 7 days), for 17-OHP when stored at -20 °C (only stable until 3 months), for cortisone at RT and 4 °C (only stable until 14 days), and cortisol at RT (only stable until 3 months)., Conclusions: Overall, we demonstrated stability of steroid hormone concentrations in DBS under various conditions which may be encountered during shipping to the diagnostic laboratory and during long-term storage before analysis., (© 2024 the author(s), published by De Gruyter, Berlin/Boston.)

Background: Subclinical thyroid diseases are often the subject of debate concerning their clinical significance, the appropriateness of diagnostic testing, and possible treatment. This systematic review addresses the variation in international guidelines for subclinical hyperthyroidism, focusing on diagnostic workup, treatment, and follow-up recommendations., Methods: Following the PRISMA guidelines, we searched PubMed, Embase, and guideline-specific databases and included clinical practice guidelines with recommendations on subclinical hyperthyroidism. Guideline recommendations were extracted, and quality assessment was performed using selected questions of the Appraisal of Guidelines for Research & Evaluation (AGREE) II instrument., Results: Of the 2624 records screened, 22 guidelines were included, which were published between 2007 and 2021. Guideline quality was generally intermediate to low. Diagnostic approaches differed substantially, particularly in the extent of recommended testing. Treatment initiation depended on TSH levels, age, and comorbidities, but the level of detail regarding defining precise comorbidities varied. Recommendations for monitoring intervals for follow-up ranged from 3 to 12 months., Conclusion: This review underscores the existing variability in (inter)national guidelines concerning subclinical hyperthyroidism. There isa need for clear recommendations in guidelines considering diagnostic workup, treatment, and follow-up of subclinical hyperthyroidism. In order to establish this, future research should focus on determining clear and evidence-based intervention thresholds.

Cryoactivation is known to occur in whole blood and plasma samples when kept between +4 and -5 °C, leading to falsely high renin concentrations. In 2022 it has been clearly shown that cryoactivation can also occur in samples stored at -20 °C. Based on these new findings, here we discuss how this can influence the clinical diagnosis of patients. First, we show that storage of renin plasma samples can affect the renin measurements and thereby the aldosterone to renin ratio (ARR) calculation, which might explain the high intraindividual variability in ARR also recently demonstrated. Second, we discuss the existing studies on the establishment of renin reference intervals and note the lack of attention given to this recently revealed preanalytical condition. Our literature review of the reference intervals for renin suggest that cryoactivation might have influenced the published data., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society.)

Background: Initial evaluation of the hypothalamus-pituitary-thyroid axis is done by measuring serum free thyroxine (fT4) and thyrotropin concentrations. For correct interpretation of these measurements, reliable age-specific reference intervals (RIs) are fundamental. Since neonatal fT4 RIs conforming to the Clinical and Laboratory Standards Institute guidelines are not available for all assays, we set out to create literature-based uniform age-specific neonatal fT4 RIs that may be used for every assay. Methods: For meta-analysis of individual participant fT4 concentrations, we systematically searched MEDLINE and Embase (search date December 6, 2023; PROSPERO registration CRD42016041871). We searched for studies reporting fT4 concentrations in healthy term newborns aged 2-27 days, born to mothers without thyroid disease in iodine-sufficient regions. Authors were invited to supply data. Due to standardization differences between assays, data could not be combined for meta-analysis directly, and we attempted to normalize the data using two distinct methods. Results: We obtained 4206 fT4 concentrations from 20 studies that used 13 different assays from 6 manufacturers. First, we set out to normalize fT4 data using the mean and standard deviation of (assay-specific) adult RIs. fT4 concentrations were transformed into Z-scores, assuming a normal distribution. Using a linear mixed-effects model (LMM), we still found a significant difference between fT4 concentration across studies ( p  < 0.001), after this normalization. As a second approach, we normalized the fT4 concentrations using data from a method/assay comparison study. We used the relationship between the Cobas assay and the other assays as a reference point to convert all values to Cobas values. However, this method also failed to produce consistent results, with significant differences between the normalized data (LMM p  < 0.001). Conclusions: We conclude that our attempts at normalizing fT4 assay results were unsuccessful. Confounders related to our unsuccessful analysis may be assay related and/or biological. These findings have significant implications for patient care, since relying on RIs from literature may result in erroneous interpretation of results. Therefore, we strongly recommend to establish local RIs for accurate interpretation of serum fT4 concentrations in neonates.

This case report describes the positive interference of the commonly used skin protective barrier cream used together with urine collection bags on the benzethonium chloride method for urine protein measurements in a 6-month-old female baby, leading to falsely elevated results. The interference was identified by both artificially mixing urine samples with this cream and comparing the results obtained using the benzethonium chloride method with those obtained using the pyrogallol red method., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Micronutrient deficiencies can develop in critically ill patients, arising from factors such as decreased intake, increased losses, drug interactions, and hypermetabolism. These deficiencies may compromise important immune functions, with potential implications for patient outcomes. Alternatively, micronutrient blood levels may become low due to inflammation-driven redistribution rather than consumption. This explorative pilot study investigates blood micronutrient concentrations during the first three weeks of ICU stay in critically ill COVID-19 patients and evaluates the impact of additional micronutrient administration. Moreover, associations between inflammation, disease severity, and micronutrient status were explored. We measured weekly concentrations of vitamins A, B6, D, and E; iron; zinc; copper; selenium; and CRP as a marker of inflammation state and the SOFA score indicating disease severity in 20 critically ill COVID-19 patients during three weeks of ICU stay. Half of the patients received additional (intravenous) micronutrient administration. Data were analyzed with linear mixed models and Pearson's correlation coefficient. High deficiency rates of vitamins A, B6, and D; zinc; and selenium (50-100%) were found at ICU admission, along with low iron status. After three weeks, vitamins B6 and D deficiencies persisted, and iron status remained low. Plasma levels of vitamins A and E, zinc, and selenium improved. No significant differences in micronutrient levels were found between patient groups. Negative correlations were identified between the CRP level and levels of vitamins A and E, iron, transferrin, zinc, and selenium. SOFA scores negatively correlated with vitamin D and selenium levels. Our findings reveal high micronutrient deficiency rates at ICU admission. Additional micronutrient administration did not enhance levels or expedite their increase. Spontaneous increases in vitamins A and E, zinc, and selenium levels were associated with inflammation resolution, suggesting that observed low levels may be attributed, at least in part, to redistribution rather than true deficiencies.

Objectives: Current liquid chromatography-tandem mass spectrometry (LC-MS/MS) applications for circulating androgen measurements are technically diverse. Previously, variable results have been reported for testosterone. Data are scarce for androstenedione and absent for dehydroepiandrosterone sulfate (DHEAS). We assessed the agreement of androstenedione, DHEAS and testosterone LC-MS/MS measurements among nine European centers and explored benefits of calibration system unification., Methods: Androgens were measured twice by laboratory-specific procedures in 78 patient samples and in EQA materials. Results were obtained by in-house and external calibration. Intra- and inter-laboratory performances were valued., Results: Intra-laboratory CVs ranged between 4.2-13.2 % for androstenedione, 1.6-10.8 % for DHEAS, and 4.3-8.7 % and 2.6-7.1 % for female and male testosterone, respectively. Bias and trueness in EQA materials were within ±20 %. Median inter-laboratory CV with in-house vs. external calibration were 12.0 vs. 9.6 % for androstenedione (p<0.001), 7.2 vs. 4.9 % for DHEAS (p<0.001), 6.4 vs. 7.6 % for female testosterone (p<0.001) and 6.8 and 7.4 % for male testosterone (p=0.111). Median bias vs. all laboratory median with in-house and external calibration were -13.3 to 20.5 % and -4.9 to 18.7 % for androstenedione, -10.9 to 4.8 % and -3.4 to 3.5 % for DHEAS, -2.7 to 6.5 % and -11.3 to 6.6 % for testosterone in females, and -7.0 to 8.5 % and -7.5 to 11.8 % for testosterone in males, respectively., Conclusions: Methods showed high intra-laboratory precision but variable bias and trueness. Inter-laboratory agreement was remarkably good. Calibration system unification improved agreement in androstenedione and DHEAS, but not in testosterone measurements. Multiple components, such as commutability of calibrators and EQA materials and internal standard choices, likely contribute to inter-laboratory variability., (© 2024 the author(s), published by De Gruyter, Berlin/Boston.)

Objectives: 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., Methods: 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., Results: 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., Conclusions: 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., (© 2024 the author(s), published by De Gruyter, Berlin/Boston.)

Background: Androgens are synthesized from cholesterol through sequential conversions by enzymes in the adrenal glands and gonads. Serum levels of androgens change during the different phases of life and regulate important developmental and maturational processes. Androgen excess or deficiency can therefore present at various ages in various ways., Content: The diagnostic approach for atypical genitalia, premature pubarche, delayed pubertal onset or progression, and hirsutism or virilization, including measurement of androgens (testosterone, androstenedione, 17-OHprogesterone, dehydroepiandrosterone, and dihydrotestosterone) is discussed in the current review. Androgens can be measured in serum, saliva, urine, or dried blood spots. Techniques to measure androgens, including immunoassays and LC-MS, have their own advantages and pitfalls. In addition, pre- and postanalytical issues are important when measuring androgens., Summary: During clinical interpretation of androgen measurements, it is important to take preanalytical circumstances, such as time of blood withdrawal, into account. As immunoassays have major drawbacks, especially in samples from women and neonates, concentrations measured using these assays should be interpreted with care. Reference intervals can only be used in relation to the measurement technique and the standardization of the assay. In the near future, new androgens will probably be added to the current repertoire to further improve the diagnosis and follow-up of androgen excess or deficiency., (© Association for Diagnostics & Laboratory Medicine 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objective: Congenital hypothyroidism (CH) is an inborn thyroid hormone (TH) deficiency mostly caused by thyroidal (primary CH) or hypothalamic/pituitary (central CH) disturbances. Most CH newborn screening (NBS) programs are thyroid-stimulating-hormone (TSH) based, thereby only detecting primary CH. The Dutch NBS is based on measuring total thyroxine (T4) from dried blood spots, aiming to detect primary and central CH at the cost of more false-positive referrals (FPRs) (positive predictive value (PPV) of 21% in 2007-2017). An artificial PPV of 26% was yielded when using a machine learning-based model on the adjusted dataset described based on the Dutch CH NBS. Recently, amino acids (AAs) and acylcarnitines (ACs) have been shown to be associated with TH concentration. We therefore aimed to investigate whether AAs and ACs measured during NBS can contribute to better performance of the CH screening in the Netherlands by using a revised machine learning-based model., Methods: Dutch NBS data between 2007 and 2017 (CH screening results, AAs and ACs) from 1079 FPRs, 515 newborns with primary (431) and central CH (84) and data from 1842 healthy controls were used. A random forest model including these data was developed., Results: The random forest model with an artificial sensitivity of 100% yielded a PPV of 48% and AUROC of 0.99. Besides T4 and TSH, tyrosine, and succinylacetone were the main parameters contributing to the model's performance., Conclusions: The PPV improved significantly (26-48%) by adding several AAs and ACs to our machine learning-based model, suggesting that adding these parameters benefits the current algorithm.

Background: Systemic inflammation plays a key role in the development of bronchopulmonary dysplasia (BPD). Cortisol is known to dampen inflammation. However, adrenal function following preterm birth is characterized by insufficient cortisol levels for the degree of inflammation, and a relative abundancy of cortisol precursors. We investigated whether this pattern could contribute to the development of BPD in preterm infants born <30 weeks of gestation., Methods: Cortisol, cortisone, 17-OH progesterone (17-OHP) and 11-deoxycortisol were measured in serum obtained at postnatal days 1, 3, 7, 14 and 28, using liquid-chromatography-tandem-mass-spectrometry. The presence of BPD was ascertained at 36 weeks postmenstrual age., Results: Sixty-five infants were included for analysis, of whom 32 (49%) developed BPD. Preterm infants developing BPD, as compared to those without BPD, had higher levels of 17-OHP, 11-deoxycortisol and cortisone relative to cortisol in their first week of life, but not at birth or beyond day 7., Conclusion: Preterm infants developing BPD had higher levels of cortisol precursors and cortisone relative to cortisol in their first week of life than infants without BPD. These findings suggest that BPD is preceded by an activated hypothalamus-pituitary-adrenal axis that could not meet the high cortisol demands, which may predispose to inflammation and BPD., Impact: Relative adrenal insufficiency is common in the first weeks after preterm birth, resulting in insufficient cortisol production for the degree of inflammation and a relative abundance of cortisol precursors; Whether this pattern contributes to the development of bronchopulmonary dysplasia (BPD) is not fully elucidated, since most studies focused on cortisol levels; Preterm infants developing BPD had higher levels of cortisol precursors and cortisone relative to cortisol in the first week of life, suggestive of a hypothalamus-pituitary-adrenal-axis activation during BPD development which cannot meet the high cortisol demands in tissues; This glucocorticoid pattern is likely to dispose to inflammation and BPD., (© 2023. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

Objective: International guidelines concerning subclinical hyperthyroidism and thyroid cancer advice absolute cut-off values for aiding clinical decisions in the low range of thyroid-stimulating hormone (TSH) concentrations. As TSH assays are known to be poorly standardized in the normal to high range, we performed a TSH assay method comparison focusing on the low range., Methods: Sixty samples, selected to cover a wide range of TSH concentrations (<0.01 to 120 mIU/L) with oversampling in the lower range (<0.4 mIU/L), were used for the method comparison between three TSH immunoassays (Cobas, Alinity and Atellica). In addition, 20 samples were used to assess the coefficient of variation from duplicate measurements in these three methods., Results: The TSH immunoassays showed standardization differences with a bias of 7-16% for the total range and 1-14% for the low range. This could lead to a different classification of 1.5% of all measured TSH concentrations <0.40 mIU/L measured in our laboratory over the last 6 months, regarding the clinically important cut-off value of TSH = 0.1 mIU/L. As the imprecision of the immunoassays varied from 1.6-5.5%, this could lead to a similar reclassification as the bias between immunoassays., Conclusions: We established the standardization differences of frequently used TSH assays for the total and low concentration ranges. Based on the proportional bias and the imprecision, this effect seems to have limited clinical consequences for the low TSH concentration range. Nevertheless, as guidelines mention absolute TSH values to guide clinical decision-making, caution must be applied when interpreting values close to these cut-offs.

Background and Aims: Graves' Disease (GD) is an autoimmune form of hyperthyroidism where autoantibodies are directed against the TSH-receptor (TSH-receptor antibodies; TRAb). GD is suspected if TRAb concentrations are above a pre-specified cut-off value. TRAb concentrations are measured using immunoassays. This study aimed to compare the performance of the recently implemented Alinity immunoassay to the KRYPTOR and Cobas TRAb immunoassays., Materials and Methods: Left-over serum samples in which TRAb concentrations were measured (KRYPTOR) were used. First, TRAb stability at -20 °C for four to six years and up to five freeze-thaw cycles were assessed. Second, TRAb measurements (n = 436) were repeated using the Alinity and Cobas immunoassay and results (scored as positive/negative based on cut-off value) were compared., Results: TRAb results were stable over five years and up to five freeze-thaw cycles. When comparing immunoassays, 86.2% of the results were similar. Total discrepancy differed between the immunoassays (5.4% Cobas vs Alinity, 8.8% Alinity vs KRYPTOR, 13.3 % Cobas vs KRYPTOR). The KRYPTOR immunoassay showed more negative TRAb results than Cobas and Alinity., Conclusion: The Alinity immunoassay showed comparable TRAb results, even though slightly more positive results compared to the KRYPTORand slightly more negative results compared to the Cobas immunoassay were seen., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Thyroid hormone (TH) is indispensable for brain development in utero and during the first 2-3 years of life, and the negative effects of TH deficiency on brain development are irreversible. Detection of TH deficiency early in life by neonatal screening allows early treatment, thereby preventing brain damage. Inborn shortage of TH, also named congenital hypothyroidism (CH), can be the result of defective thyroid gland development or TH synthesis (primary or thyroidal CH (CH-T)). Primary CH is characterized by low blood TH and elevated thyroid-stimulating hormone (TSH) concentrations. Less frequently, CH is due to insufficient stimulation of the thyroid gland because of disturbed hypothalamic or pituitary function (central CH). Central CH is characterized by low TH concentrations, while TSH is normal, low or slightly elevated. Most newborn screening (NBS) programs for CH are primarily TSH based and thereby do not detect central CH. Only a few NBS programs worldwide aim to detect both forms of CH by different strategies. In the Netherlands, we have a unique T4-TSH-thyroxine-binding globulin (TBG) NBS algorithm for CH, which enables the detection of primary and central CH. Although the necessity of central CH detection by NBS is still under debate, it has been shown that most central CH patients have moderate-to-severe hypothyroidism instead of mild and that early detection of central CH by NBS probably improves its clinical outcome and clinical care for central CH patients with multiple pituitary hormone deficiency. We are therefore convinced that detection of central CH by NBS is of utmost importance.

Hormonal contraceptives (HC) are widely used among women in reproductive ages. In this review, the effects of HCs on 91 routine chemistry tests, metabolic tests, and tests for liver function, hemostatic system, renal function, hormones, vitamins and minerals were evaluated. Test parameters were differently affected by the dosage, duration, composition of HCs and route of administration. Most studies concerned the effects of combined oral contraceptives (COC) on the metabolic, hemostatic and (sex) steroids test results. Although the majority of the effects were minor, a major increase was seen in angiotensinogen levels (90-375 %) and the concentrations of the binding proteins (SHBG [∼200 %], CBG [∼100 %], TBG [∼90 %], VDBP [∼30 %], and IGFBPs [∼40 %]). Also, there were significant changes in levels of their bound molecules (testosterone, T3, T4, cortisol, vitamin D, IGF1 and GH). Data about the effects of all kinds of HCs on all test results are limited and sometimes inconclusive due to the large variety in HC, administration routes and dosages. Still, it can be concluded that HC use in women mainly stimulates the liver production of binding proteins. All biochemical test results of women using HC should be assessed carefully and unexpected test results should be further evaluated for both methodological and pre-analytical reasons. As HCs change over time, future studies are needed to learn more about the effects of other types, routes and combinations of HCs on clinical chemistry tests., (© 2023 the author(s), published by De Gruyter, Berlin/Boston.)

Research Question: What is the risk of hypogonadism in men with obstructive azoospermia, non-obstructive azoospermia (NOA) or Klinefelter syndrome after testicular sperm extraction (TESE)?, Design: This prospective longitudinal cohort study was carried out between 2007 and 2015., Results: Around 36% of men with Klinefelter syndrome, 4% of men with obstructive azoospermia and 3% of men with NOA needed testosterone replacement therapy (TRT). Klinefelter syndrome was strongly associated with TRT while no association was found between obstructive azoospermia or NOA and TRT. Irrespective of the pre-operative diagnosis, a higher testosterone concentration before TESE was associated with a lower chance of needing TRT., Conclusions: Men with obstructive azoospermia or NOA have a similar moderate risk of clinical hypogonadism after TESE, while this risk is much larger for men with Klinefelter syndrome. The risk of clinical hypogonadism is lower when testosterone concentrations are high before TESE., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objective: The Dutch Congenital hypothyroidism (CH) Newborn Screening (NBS) algorithm for thyroidal and central congenital hypothyroidism (CH-T and CH-C, respectively) is primarily based on determination of thyroxine (T4) concentrations in dried blood spots, followed by thyroid-stimulating hormone (TSH) and thyroxine-binding globulin (TBG) measurements enabling detection of both CH-T and CH-C, with a positive predictive value (PPV) of 21%. A calculated T4/TBG ratio serves as an indirect measure for free T4. The aim of this study is to investigate whether machine learning techniques can help to improve the PPV of the algorithm without missing the positive cases that should have been detected with the current algorithm., Design & Methods: NBS data and parameters of CH patients and false-positive referrals in the period 2007-2017 and of a healthy reference population were included in the study. A random forest model was trained and tested using a stratified split and improved using synthetic minority oversampling technique (SMOTE). NBS data of 4668 newborns were included, containing 458 CH-T and 82 CH-C patients, 2332 false-positive referrals and 1670 healthy newborns., Results: Variables determining identification of CH were (in order of importance) TSH, T4/TBG ratio, gestational age, TBG, T4 and age at NBS sampling. In a Receiver-Operating Characteristic (ROC) analysis on the test set, current sensitivity could be maintained, while increasing the PPV to 26%., Conclusions: Machine learning techniques have the potential to improve the PPV of the Dutch CH NBS. However, improved detection of currently missed cases is only possible with new, better predictors of especially CH-C and a better registration and inclusion of these cases in future models., Competing Interests: Declaration of Competing Interest Annet M. Bosch has received a speaker’s fee from Nutricia and has been a member of advisory boards for Biomarin., (Copyright © 2023. Published by Elsevier Inc.)

Objective: Interpreting laboratory results for transgender individuals who started hormone therapy requires careful consideration, specifically for analytes that have sex-specific reference intervals. In literature, conflicting data exist on the effect of hormone therapy on laboratory parameters. By studying a large cohort, we aim to define what reference category (male or female) is most appropriate to use for the transgender population over the course of gender-affirming therapy., Methods: A total of 2201 people (1178 transgender women and 1023 transgender men) were included in this study. We analyzed hemoglobin (Hb), hematocrit (Ht), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT), creatinine, and prolactin, at three different time points: pretreatment, during hormone therapy, and after gonadectomy., Results: For transgender women, Hb and Ht levels decrease after initiation of hormone therapy. The concentration of liver enzymes ALT, AST, and ALP decrease whereas the levels of GGT do not change statistically significantly. Creatinine levels decrease whereas prolactin levels rise in transgender women during gender-affirming therapy. For transgender men Hb and Ht values increase after starting hormone therapy. Liver enzymes and creatinine levels increase statistically significant as well upon hormone therapy while prolactin concentrations decrease. Overall, reference intervals in transgender people after 1 year on hormone therapy resembled those of their affirmed gender., Conclusions: Generating transgender-specific reference intervals is not essential to correctly interpret laboratory results. As a practical approach, we recommend to use the reference intervals of the affirmed gender from 1 year onwards after starting hormone therapy., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Society of Endocrinology.)

Objective: This work aims to review and discuss controversial topics in the field of vitamin D, SARS-CoV-2 infection, and COVID-19., Methods: The International Conferences "Controversies in Vitamin D" are a series of workshops that started in 2017 featuring international experts and leaders in vitamin D research and clinical practice. The fifth annual conference was held in Stresa, Italy, September 15 to 18, 2021., Evidence: Before the event, participants reviewed available studies on their assigned topic, drafted a related abstract, and presented their findings at the time of the conference. Relevant literature that became available since was also discussed within the panel and updated accordingly., Consensus: Before the event, the drafted abstracts had been merged to prepare a preliminary document. After the conference presentations, in-depth discussions in open sessions led to consensus. The document was subsequently modified according to discussions and up-to-date literature inclusion., Conclusions: There is quite consistent evidence for an association between low 25 OH vitamin D (25(OH)D) levels and poor COVID-19 outcomes, despite heterogeneous publications of variable quality. However, the low vitamin D status in COVID-19 patients might also reflect reverse causality. Vitamin D supplementation might have a positive role in COVID-19 prevention. The evidence supporting a beneficial effect of vitamin D treatment in decreasing the risk of COVID-19 complications is conflicting. Conclusive statements regarding the beneficial effect of vitamin D in this context await high-quality, randomized controlled trials., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: The exploration of the metabolites in the degradation pathways of vitamin D (VTD) has gained importance in recent years and simultaneous quantitation of twenty-five-hydroxy vitamin D (25(OH)D) mass concentration together with 24,25-dihydroxyvitamin D (24,25(OH)2D) has been proposed as a newer approach to define VTD deficiency. Yet, no data are available on 24,25(OH)2D biological variation (BV). In this study, we evaluated 24,25(OH)2D's BV on the European Biological Variation Study (EuBIVAS) cohort samples to determine if analytical performance specifications (APS) for 24,25(OH)2D could be generated., Methods: Six European laboratories recruited 91 healthy participants. 25(OH)D and 24,25(OH)2D concentrations in K 3 -EDTA plasma were examined weekly for up to 10 weeks in duplicate with a validated LC-MS/MS method. The Vitamin D Metabolite Ratio (24,25(OH)2D divided by 25(OH)D × 100) was also calculated at each time point., Results: Linear regression of the mean 24,25(OH)2D concentrations at each blood collection showed participants were not in steady state. Variations of 24,25(OH)2D over time were significantly positively associated with the slopes of 25(OH)D concentrations over time and the concentration of 25(OH)D of the participant at inclusion, and negatively associated with body mass index (BMI), but not with age, gender, or location of the participant. The variation of the 24,25(OH)2D concentration in participants over a 10 weeks period was 34.6%. Methods that would detect a significant change linked to the natural production of 24,25(OH)2D over this period at p<0.05 would need a relative measurement uncertainty ( u %)<14.9% while at p<0.01, relative measurement uncertainty should be <10.5%., Conclusions: We have defined for the first time APS for 24,25(OH)2D examinations. According to the growing interest in this metabolite, several laboratories and manufacturers might aim to develop specific methods for its determination. The results presented in this paper are thus necessary prerequisites for the validation of such methods., (© 2023 the author(s), published by De Gruyter, Berlin/Boston.)

Objectives: Free thyroxine (FT4) in serum is routinely measured in clinical practice to diagnose and monitor thyroid disease. Due to its concentration in picomolar range and the delicate equilibrium of free and protein-bound T4, accurate measurement is challenging. As a consequence, large inter-method differences in FT4 results exists. Optimal method design and standardization of the FT4 measurement is therefore necessary. The IFCC Working Group for Standardization of Thyroid Function Tests proposed a reference system with a conventional reference measurement procedure (cRMP) for FT4 in serum. In this study, we describe our FT4 candidate cRMP and its validation in clinical samples., Methods: This candidate cRMP is based on equilibrium dialysis (ED) combined with determination of T4 with an isotope-dilution liquid chromatography tandem mass-spectrometry (ID-LC-MS/MS) procedure and was developed according to the endorsed conventions. Its accuracy, reliability, and comparability was investigated using human sera., Results: It was shown that the candidate cRMP adhered to the conventions and its accuracy, precision, and robustness were adequate in serum of healthy volunteers., Conclusions: Our candidate cRMP measures FT4 accurately and performs well in serum matrix., (© 2023 the author(s), published by De Gruyter, Berlin/Boston.)

Introduction: In our hospital, physicians noticed high free thyroxine (fT4) concentrations without complete suppression of thyroid-stimulating hormone (TSH) in blood samples of patients at the outpatient clinic, which appeared to occur more often following the introduction of a new fT4 immunoassay. This discordance may be explained by incorrect reference intervals, analytical issues, or patient-related factors. We aimed to establish the contribution of the possible factors involved., Methods: Reference intervals of both fT4 immunoassays were re-evaluated using blood samples of healthy volunteers and the new immunoassay's performance was assessed using internal quality controls and external quality rounds. The frequency of discordant fT4 and TSH pairings obtained from laboratory requests were retrospectively analysed using a Delfia (n = 3174) and Cobas cohort (n = 3408). Last, a literature search assessed whether the time of blood draw and the time of levothyroxine (L-T4) ingestion may contribute to higher fT4 concentrations in L-T4 users., Results: The original reference intervals of both fT4 immunoassays were confirmed and no evidence for analytical problems was found. The Delfia (n = 176, 5.5%) and Cobas cohorts (n = 295, 8.7%) showed comparable frequencies of discordance. Interestingly, 72-81% of the discordant results belonged to L-T4 users. Literature indicated the time of blood withdrawal of L-T4 users and, therefore, the time of L-T4 intake as possible explanations., Conclusions: High fT4 without suppressed TSH concentrations can mainly be explained by L-T4 intake. Physicians and laboratory specialists should be aware of this phenomenon to avoid questioning the assay's performance or unnecessarily adapting the L-T4 dose in patients.

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.

Objectives: Hormone measurements using automated immunoassays (IAs) can be affected by the sample matrix. Liquid chromatography tandem-mass spectrometry (LC-MS/MS) is less affected by these matrix effects. In clinical laboratories, testosterone, cortisol and, free thyroxine (FT4) are often measured using IAs. Renal failure alters serum composition in blood samples from people undergoing hemodialysis (HDp) and have, therefore, a complex serum constitution compared to healthy controls (HC). The goal of this study was to investigate the accuracy of testosterone, cortisol, and FT4 measurements in samples of HDp and to get more insight in the interfering factors., Methods: Thirty serum samples from HDp and HC were collected to measure testosterone, cortisol, and FT4 using a well standardized isotope dilution (ID)-LC-MS/MS method and 5 commercially available automated IAs (Alinity, Atellica, Cobas, Lumipulse, UniCel DXI). Method comparisons between LC-MS/MS and IAs were performed using both HDp and HC samples., Results: Average bias from the LC-MS/MS was for testosterone, cortisol, and FT4 immunoassays respectively up to 92, 7-47 and 16-27% more in HDp than in HC samples and was IA dependent. FT4 IA results were falsely decreased in HDp samples, whereas cortisol and testosterone concentrations in females were predominantly falsely increased. Correlation coefficients between LC-MS/MS and IA results were lower in HDp compared to HC samples., Conclusions: Several IAs for testosterone (in women), cortisol, and FT4 are less reliable in the altered serum matrix of samples of HDp than in HC. Medical and laboratory specialists should be aware of these pitfalls in this specific population., (© 2023 the author(s), published by De Gruyter, Berlin/Boston.)

Common symptoms of overt hypothyroidism are non-specific and include fatigue, lethargy, and dry skin. Although the diagnosis is considered to be straightforward, no single symptom can be used to identify patients with overt hypothyroidism, while many patients with subclinical hypothyroidism are asymptomatic. A large population-based study on the spectrum of symptoms in subclinical hypothyroidism showed similar rates of thyroid disease-related symptoms compared with euthyroid subjects, while the TSH concentration had no impact on symptom score. Together, these findings make it challenging to attribute symptoms to their underlying cause. This is also true in the case of unexplained persistent symptoms in levothyroxine-treated patients. Although generally considered a life-long replacement therapy, successful thyroid hormone discontinuation resulting in euthyroidism has been reported in approximately one third of patients. Thus, we overtreat patients with (subclinical) hypothyroidism, highlighting the importance of reliable diagnostic criteria. The diagnostic process, including the implementation of robust TSH and FT4 reference intervals, is especially challenging in specific situations including aging, pregnancy, non-thyroidal illness, and central hypothyroidism. There is a clear need for improved adherence to current guidelines from scientific societies and for willingness to manage symptoms without a clear pathological correlate, especially in the case of mild TSH elevations. This review will highlight recent literature on this topic and offers some practice points., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Jansen, Boelen, Heijboer, Bruinstroop and Fliers.)

Two decades after the discovery of the hormone FGF23, we know more about phosphate homeostasis as it turned out that FGF23 is the central hormone that regulates this. Hereditary hypophosphatemic rickets and tumor-induced osteomalacia could by then be explained, by autonomous FGF23 production, and the nephrology field was excited by this new marker as it turned out to be independently associated with mortality in people treated by hemodialysis. This led to the development of several immunoassays to be able to measure FGF23 in blood. In the past years we learned that FGF23 is a rather stable peptide, the precision of the assays is acceptable but assays are not standardized and therefore not comparable. This means that reference values and cutoff values need to be assay specific. For several assays reference values have been established and gender and age did not seem of high importance. The phosphate content of the diet, which can be culturally dependent, however, should be taken into account when interpreting results, but to what extent is not totally clear. Currently, clinical application of the immunoassays is established in the diagnosis of hereditary hypophosphatemic rickets and diagnosis and follow-up of tumor-induced osteomalacia. Definite conclusions on the usefulness of the FGF23 measurement in people with CKD either as a marker for risk prediction or a as target for treatment remains to be determined. The latter applications would require dedicated prospective clinical trials, which may take years, before providing answers. To improve the standardization of the FGF23 assays and to shed light on the biological functions that fragments might have we might aim for an LC-MS/MS-based method to quantify both intact and fragmented FGF23. In this literature review we will summarize the current knowledge on the physiological role of FGF23, its quantification, and the clinical usefulness of its determination., (© 2022. The Author(s).)