Stability of steroid hormones in dried blood spots (DBS).

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.

The effect of hormonal contraceptive therapy on clinical laboratory parameters: a literature review.

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.

Neonatal reference intervals for serum steroid hormone concentrations measured by LC-MS/MS.

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.

Should we depend on reference intervals from manufacturer package inserts? Comparing TSH and FT4 reference intervals from four manufacturers with results from modern indirect methods and the direct method.

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.

Basal cortisol in relation to metyrapone confirmation in predicting adrenal insufficiency after pituitary surgery.

PURPOSE: Pituitary surgery can lead to post-surgical adrenal insufficiency with the need for glucocorticoid replacement and significant disease related burden. In patients who do not receive hydrocortisone replacement before surgery, at our center, an early morning plasma cortisol concentration using a cut-off value of 450 nmol/L 3 days after surgery (POD3) is used to guide the need for hydrocortisone replacement until dynamic confirmatory testing using metyrapone. The aim of this study was to critically assess the currently used diagnostic and treatment algorithm in patients undergoing pituitary surgery in our pituitary reference center. METHODS: Retrospective analysis of all patients with a POD3 plasma cortisol concentration < 450 nmol/L who received hydrocortisone replacement and a metyrapone test after 3 months. Plasma cortisol concentration was measured using an electrochemiluminescence immunoassay (Roche). All patients who underwent postoperative testing using metyrapone at Amsterdam UMC between January 2018 and February 2022 were included. Patients with Cushing's disease or those with hydrocortisone replacement prior to surgery were excluded. RESULTS: Ninety-five patients were included in the analysis. The postoperative cortisol concentration above which no patient had adrenal insufficiency (i.e. 11-deoxycortisol > 200 nmol/L) was 357 nmol/L (Sensitivity 100%, Specificity 31%, PPV:32%, NPV:100%). This translates into a 28% reduction in the need for hydrocortisone replacement compared with the presently used cortisol cut-off value of 450 nmol/L. CONCLUSION: Early morning plasma cortisol cut-off values lower than 450 nmol/L can safely be used to guide the need for hydrocortisone replacement after pituitary surgery.

Lower accuracy of testosterone, cortisol, and free T4 measurements using automated immunoassays in people undergoing hemodialysis.

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.

Instability of corticotropin during long-term storage - myth or reality?

OBJECTIVES: Corticotropin is notorious for its instability. Whereas several studies have investigated its short-term stability in plasma following venous blood sampling, studies on long-term stability are lacking. Here we investigated the long-term storage stability of corticotropin in ethylenediaminetetraacetic acid containing plasma. METHODS: Specimens from healthy volunteers (neat, spiked) were stored in polypropylene microcentrifuge tubes with socket screw-caps at -20 °C and -70 °C for up to one and a half years. Corticotropin in plasma was measured using an Abbott research only immunoassay. Separately, specimens from patients were collected during diagnostic routine testing and stored in polystyrene tubes with push-caps at -20 °C for up to 6 years. In these samples corticotropin hormone was measured using the Diasorin corticotropin immunoassay. RESULTS: Storage of specimens at -20 °C or -70 °C for up to one and a half years showed minimal changes (<11%) in corticotropin levels, while storage of patient samples at -20 °C for up to 6 years showed a significant (54%) reduction in corticotropin levels. CONCLUSIONS: Corticotropin levels are stable in plasma when stored at -20 °C for one and a half years using the Abbott research only assay, but with longer storage time a significant reduction in corticotropin levels can be expected. Once specimens are stored for future corticotropin measurements, one should consider storage time, storage temperature and assay differences.

Oxidation of PTH: in vivo feature or effect of preanalytical conditions?

BACKGROUND: Posttranslational oxidation of parathyroid hormone (PTH) modifies its biological activity. Measurement of non-oxidized PTH (n-oxPTH) could be an improvement in assessing PTH status, as intact PTH may rather reflect oxidative stress. However, it is debated whether oxidation of PTH occurs in vivo, or whether it is mainly an in vitro artifact. The aim of this study was to investigate the influence of different preanalytical conditions on the oxidation of PTH within a wide range of plasma PTH concentrations and oxidation propensity. METHODS: n-oxPTH was separated from its oxidized form using an affinity column capturing the oxidized PTH. n-oxPTH was measured in eluate using commercially available PTH assays. The study included ethylenediaminetetraacetic acid plasma samples from 17 patients undergoing hemodialysis and 32 healthy subjects. We determined effects of storage temperature, time until centrifugation and freeze-thaw cycles. PTH and n-oxPTH concentrations were measured in each sample using six different immunoassays. RESULTS: n-oxPTH concentrations remained unchanged up to 180 min until centrifugation, two freeze-thaw cycles or after storage at -20°C or -80°C up to 79 days. Various methods for n-oxPTH and PTH measurements yielded highly comparable results, apart from standardization differences between various PTH and n-oxPTH assays. CONCLUSIONS: n-oxPTH concentrations were stable under our study conditions, indicating negligible ex vivo oxidation of PTH. In addition, PTH immunoassays have a different sensitivity for n-oxPTH than for total PTH. For this reason, the n-oxPTH/total PTH ratio cannot be used in absence of a n-oxPTH standard. Clinical implications of determining n-oxPTH require additional study.

Thyroglobulin and calcitonin measurements: pitfalls and future perspectives.

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.

The Clinical Impact of Sample Storage at -20†°C on Renin Reference Intervals and Aldosterone-Renin Ratio Calculations.

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.

Approach to the Patient: Challenging Cases of Pediatric Thyrotoxicosis.

Graves' disease (GD) is the leading cause of hyperthyroidism in children. However, compared to adults GD in children is a rare condition. In a recent guideline issued by the European Thyroid Association the diagnostic evaluation and treatment of pediatric GD is described extensively. In this article we go beyond the guideline and describe the potential challenges of establishing the right etiology of thyrotoxicosis in children, illustrated by cases of thyroid hormone resistance, autonomous functioning thyroid nodules and subacute thyroiditis with a thyrotoxic phase. In addition, we report therapeutic challenges in pediatric GD such as recurrent immunological flare-ups under anti-thyroid drug (ATD) treatment, innovative ways to improve ATD compliance and the role of definitive treatment in persistent complaints of malaise under ATD treatment.

Confusion in the interpretation of prolactin levels caused by inappropriately low reference intervals.

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 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 our the Amsterdam UMC hospital. Prolactin levels were measured using the AtellicaTM IM analyzer (Siemens Healthineers) and the Cobas (Roche Diagnostics) immunoassay. Reference intervals 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 reference intervals 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 reference intervals are different for the Atellica and Cobas assays. We recommend that laboratory specialists and manufacturers periodically review prolactin assay RIs as incorrect reference intervals can lead to misinterpretation of prolactin levels and unnecessary referrals and further laboratory testing, as we have experienced.

Age-Specific Reference Intervals for Thyroid-Stimulating Hormones and Free Thyroxine to Optimize Diagnosis of Thyroid Disease.

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.

How low can we (reliably) go? A method comparison of thyroid-stimulating hormone assays with a focus on low concentrations.

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.

Stability of TSH receptor antibody concentrations and comparability of its immunoassays.

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.

Increased fT4 concentrations in patients using levothyroxine without complete suppression of TSH.

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.

Mandometer treatment not superior to treatment as usual for anorexia nervosa.

OBJECTIVE: A comparison of the efficacy of a novel treatment method for anorexia nervosa (AN), the Mandometer treatment (MT), with treatment as usual (TAU). METHOD: During treatment data were collected to determine weight recovery and outcome as assessed by the Morgan Russell Outcome Assessment Schedule (MROAS). RESULTS: After treatment 63% of the MT group and 85% of the TAU group had reached a normal weight level and both MT and TAU showed a good outcome on the MROAS (75 and 71%, respectively). After two years more MT than TAU patients were still in treatment and more MT patients had relapsed. DISCUSSION: The outcome for both treatments in our study were similar and comparable with, if not better than outcome data of other AN studies. MT is not superior to TAU in outcome results and in relapse rate during the first two years following admission for AN treatment.

Pregnancy disrupts the accuracy of automated fT4 immunoassays.

Objective: Thyroid hormone measurements are often performed in pregnant women, as hypo- and hyperthyroidism during pregnancy can severely affect the fetus. Serum free thyroxine (fT4) measurements are well known for their analytical challenges, due to low serum concentrations and the subtle equilibrium between free and bound T4 (to thyroid-binding globulin (TBG), transthyretin and albumin). Pregnant women have high TBG concentrations due to an increase in human chorionic gonadotropin (hCG) and estrogen and lower albumin concentrations which change the equilibrium and may affect the validity of fT4 measurements in their samples. As accurate serum fT4 measurements in pregnant women are important for the long-term health of the fetus, we aimed to evaluate the accuracy of several fT4 immunoassays in the serum of pregnant women. Methods: FT4 was measured in healthy controls and pregnant women using a candidate-reference method (LC-MS/MS) and five commercially available automated immunoassays (Alinity (Abbott), Atellica (Siemens), Cobas (Roche), Lumipulse (Fujirebio) and UniCel DXI (Beckman Coulter)). Method comparisons (Bland Altman plots and Passing and Bablok analyses) were performed. Results: Serum samples from both healthy controls (n = 30) and pregnant women (n = 30; mean gestational age, 24.8 weeks) were collected. The fT4 immunoassays deviated +7 to +29% more from the LC-MS/MS in serum samples of pregnant women than healthy controls (falsely high). Conclusions: Our results indicate that immunoassays overestimate fT4 in pregnant women, which might lead to an overestimation of thyroid status. Physicians and laboratory specialists should be aware of this phenomenon to avoid drawing false conclusions about thyroid function in pregnant women.

Identification of a Novel CYP11B2 Variant in a Family with Varying Degrees of Aldosterone Synthase Deficiency.

Isolated aldosterone synthase deficiency is a rare autosomal recessive disorder caused by pathogenic variants in CYP11B2, resulting in impaired aldosterone synthesis. We report on a neonate with isolated aldosterone synthase deficiency caused by a novel homozygous CYP11B2 variant Chr8:NM_000498.3:c.400G>A p.(Gly134Arg). The patient presented shortly after birth with severe signs of aldosterone deficiency. Interestingly, segregation analysis revealed that the patient's asymptomatic father was also homozygous for the CYP11B2 variant. Biochemical evaluation of the father indicated subclinical enzyme impairment, characterized by elevated aldosterone precursors. Apparently, this homozygous variant led to different clinical phenotypes in two affected relatives. In this manuscript we elaborate on the performed biochemical and genetic work-up and describe potential pitfalls of CYP11B2 sequencing due to its homology to CYP11B2.

Improving Science by Overcoming Laboratory Pitfalls With Hormone Measurements.

Despite all the effort taken, there is often surprisingly little attention paid to the hormone analyses involved in research studies. Thinking carefully about the quality of the hormone measurements in these studies is, however, of major importance, as this attention to methods may prevent false conclusions and inappropriate follow-up studies. We discuss issues regarding hormone measurements that one should consider, ideally prior to starting, or otherwise, as they arise during a scientific study: quality of the technique, expertise, matrices, timing and storage conditions, freeze-thaw cycles, lot-to-lot and day-to-day variation, analyses per batch or sample-wise, singlicate or duplicate measurements, combining methods, and standardization. This article and the examples mentioned herein aim to clarify the need to pay attention to the hormone analyses, and to help in making decisions. In addition, these examples help editors and reviewers of scientific journals to pay attention to the methods section in the submitted manuscripts and ask the right critical questions when needed.