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 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.

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.

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.

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.

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.

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.

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.

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.

Defective Levothyroxine Response in a Patient with Dyshormonogenic Congenital Hypothyroidism Caused by a Concurrent Pathogenic Variant in Thyroid Hormone Receptor-ß.

Background: Pituitary resistance to thyroid hormone (PRTH) is often seen in congenital hypothyroidism (CH), presenting as elevated thyrotropin (TSH) values despite (high-)normal thyroid hormone (TH) values achieved by levothyroxine treatment. In this study, we describe a girl with CH who was referred because of difficulties interpreting thyroid function tests. She was thought to have PRTH associated with CH, but genetic studies discovered a pathogenic variant in THRB, causing resistance to TH (RTH-ß). Methods: Clinical, genetic, and biochemical data of the proband's family were collected. Results: The 3-year-old girl was diagnosed with CH due to a homozygous pathogenic c.470del p.(Asn157Thrfs*3) SLC5A5 variant in the neonatal period. She needed a notably high levothyroxine dose to normalize TSH, leading to high free thyroxine levels. There were no signs of hyperthyroidism. Sequencing identified a heterozygous pathogenic c.947G>A p.(Arg316His) THRB variant. Conclusions: To our knowledge, this is the first report of concomitant SLC5A5 and THRB variants causing CH and RTH-ß.

A Newborn Falsely Suspected of Congenital Hypothyroidism due to Mutated Thyroxine-Binding Globulin with Low Binding Affinity.

INTRODUCTION: Neonatal screening programs for congenital hypothyroidism (CH) have been implemented worldwide to facilitate early diagnosis and treatment. The Dutch neonatal CH screening is primarily based on the measurement of thyroxine (T4). When T4 is low, an additional thyroxine-binding globulin (TBG) measurement is performed to reduce the number of false-positive screening results due to harmless TBG deficiency. Here, we present a case of a rare functional TBG deficiency leading to a false suspicion of CH. CASE PRESENTATION: Neonatal screening in this patient revealed a decreased T4, normal TSH, and normal TBG concentration, suggesting central CH. However, free T4 was normal. DNA sequencing analysis revealed a novel, hemizygous mutation (c.139G>A) in SERPINA7, the gene encoding TBG, resulting in the substitution of the conserved amino acid alanine to threonine at position 27. Crystal structure analyses showed that this substitution has a detrimental effect on binding of T4 to TBG. CONCLUSIONS: The novel SERPINA7 variant in this patient led to a false suspicion of central hypothyroidism in the Dutch T4-based neonatal screening program. It is important to recognize patients with such TBG defects to prevent unnecessary additional testing and treatment.

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.

Estradiol reference intervals in women during the menstrual cycle, postmenopausal women and men using an LC-MS/MS method.

BACKGROUND: For optimal medical decision-making, harmonized reference intervals for estradiol for different ages and both sexes are needed. Our aim was to establish reference intervals using a highly accurate and traceable LC-MS/MS method and to compare these with reference intervals in literature. METHODS: Estradiol was measured in serum obtained daily during the menstrual cycle of 30 healthy premenopausal women and in serum of 64 men and 33 postmenopausal women. The accuracy of our LC-MS/MS method was demonstrated by a method comparison with the CDC reference method. RESULTS: Our LC-MS/MS method was traceable to the reference method. Estradiol reference interval during the early follicular phase (days -15 to -6) was 31-771 pmol/L; during the late follicular phase (days -5 to -1) 104-1742 pmol/L; during the LH peak (day 0) 275-2864 pmol/L; during the early luteal phase (days +1 to +4) 95-1188 pmol/L; during mid luteal phase (days +5 to +9) 151-1941 pmol/L; during late luteal phase (days +10 to +14) 39-1769 pmol/L. The reference interval for men was 12-136 pmol/L and for postmenopausal women <26 pmol/L. CONCLUSIONS: The established estradiol reference intervals can be used for all traceable LC-MS/MS methods for medical-decision making.

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.

[Specific IgE testing in food allergies]. / Specifiek-IgE-bepaling bij voedselallergie.

The interpretation of specific IgE test results in patients with suspected food allergies is not always straightforward. In fact, specific IgE test results for food allergens can provide physicians with more questions than answers. Based on the description of two patient cases, we discuss the interpretation of specific IgE test results for food allergens; the description includes the diagnostic process, cross-reactivity and component-resolved diagnostics.

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.

Acute and chronic suppression of the central ghrelin signaling system reveals a role in food anticipatory activity.

Using the rodent activity-based anorexia (ABA) model that mimics clinical features of anorexia nervosa that include food restriction-induced hyperlocomotion, we found that plasma ghrelin levels are highly associated with food anticipatory behaviour, measured by running wheel activity in rats. Furthermore, we showed that ghrelin receptor (GHS-R1A) knockout mice do not anticipate food when exposed to the ABA model, unlike their wild type littermate controls. Likewise, food anticipatory activity in the ABA model was suppressed by a GHS-R1A antagonist administered either by acute central (ICV) injection to rats or by chronic peripheral treatment to mice. Interestingly, the GHS-R1A antagonist did not alter food intake in any of these models. Therefore, we hypothesize that suppression of the central ghrelin signaling system via GHS-R1A provides an interesting therapeutic target to treat hyperactivity in patients suffering from anorexia nervosa.