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.

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.

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.

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.

Medullary thyroid cancer (MTC) originates from parafollicular cells of the thyroid gland. These cells secrete calcitonin, which can be measured in serum using immunoassays to monitor MTC. The stability of calcitonin is not well understood and may affect serum calcitonin measurements. In addition, calcitonin immunoassays are poorly standardised, hindering comparison of calcitonin concentrations between assays. Here we aimed to gain insight in the stability of calcitonin in serum and in differences between calcitonin immunoassays. The stability of calcitonin in serum was determined upon storage at room temperature for 5 h and upon five freeze-thaw cycles using the Liaison XL (Diasorin). Calcitonin method comparisons were performed using three immunoassays: Liaison XL, Cobas e601 (Roche Diagnostics) and Atellica IM 1300 (Siemens Healthineers). Changes in calcitonin concentrations in serum remain within the maximum allowable change criteria for up to 5 h at room temperature and five freeze-thaw cycles. Calcitonin method comparison showed good agreement between the Cobas e601 and Liaison XL (Calcitonin_Cobase601 = 1.02 Calcitonin_LiaisonXL-1.64) assay. The Atellica IM 1300 assay agreed less with the Cobas e601 and Liaison XL assay (Calcitonin_{AtellicaIM1300} = 1.13Calcitonin_Cobase601 + 2.59, Calcitonin_{AtellicaIM1300} = 1.13Calcitonin_LiaisonXL + 1.19). Calcitonin in serum remains stable for 5 h at room temperature and upon five freeze-thaw cycles. The method comparison between the Cobas e601 and Liaison XL showed good agreement, whereas the Atellica IM 1300 assay resulted in 13% higher concentrations than both Cobas e601 and Liaison XL. This highlights the importance of using the same immunoassay to monitor calcitonin concentrations in patients with MTC.