Cardiac troponin (cTn) testing plays a crucial role in the diagnosis of cardiovascular diseases, particularly acute coronary syndrome (ACS), which includes acute myocardial infarction (AMI). However, conventional immunoassays may be subject to interference from autoantibodies, cross-reactivity, and biotin-related effects, compromising diagnostic accuracy. A thorough investigation of these interference mechanisms is necessary to improve assay methodologies, ensuring greater reliability and precision. In recent years, significant advancements in mass spectrometry (MS) technology have sparked increased interest in its application for cTn testing. For instance, liquid chromatography-tandem mass spectrometry (LC-MS/MS) employs multiple reaction monitoring (MRM) to accurately quantify cardiac troponin I (cTnI)-specific tryptic peptides along with their fragment ions. This technique effectively reduces immunoassay interference while improving analytical specificity. Compared to traditional immunoassays, MS-based approaches alleviate matrix effects and analytical interferences while achieving superior specificity. Nonetheless, clinical adoption remains constrained by technical complexity; thus clinicians can obtain more reliable diagnostic insights. This review summarizes the current landscape of cTn detection technologies by examining the prevalence of false-positive results across various methods. It further explores both the practical applications and challenges associated with MS-based techniques in cTn testing. Ultimately, this review aims to improve cTn testing reliability, enhance cardiovascular disease diagnosis, and guide personalized treatment strategies.

Therapeutic drug monitoring is recommended to optimize infliximab use and improve outcome in chronic inflammatory disorders.

To describe a simple and affordable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to measure infliximab in serum.

Infliximab was measured using winged stable isotope-labeled peptides as internal standards. Linearity, lower limit of measuring interval, limit of detection, precision, accuracy, carryover, and ion suppression were evaluated. Method comparison against 2 enzyme-linked immunosorbent assay (ELISA) methods (Remsima Monitor and IDKmonitor Infliximab) and anti-drug antibody (ADA) interference were evaluated using clinical specimens from inflammatory bowel disease patients (N = 237).

Analytical run time and sample preparation time were 5 minutes per sample and 3 hours per batch, respectively. Analytical measurement interval and limit of detection were 0.50 to 50.0 μg/mL (R2 = 0.998) and 0.25 μg/mL, respectively. The intraday and interday imprecision percentage coefficients of variation were less than 6.1%. Accuracy was 94.2% to 98.7%. No significant ion suppression or carryover was observed. Infliximab concentrations measured by LC-MS/MS showed good agreement with those measured by Remsima Monitor (mean percentage difference, 5.7%; 95% CI, -1.2% to 12.6%) but were markedly lower than those measured by IDKmonitor (-32.6%; -35.8% to -29.4%), demonstrating significant bias between ELISAs. Although a good agreement between LC-MS/MS and ELISA was observed for ADA-negative samples (-3.5%; -12.8% to 5.9%), a significant bias was observed for ADA-positive samples (13.6%; 1.7% to 25.6%).

This simple, fast, and affordable LC-MS/MS method for infliximab quantitation could improve standardization of infliximab quantitation and optimization of infliximab use in patients with high-titer ADA.

Wildlife scientists are quantifying steroid hormones in a growing number of tissues and employing novel methods that must undergo validation before application. This study tested the accuracy and precision of liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for use on blubber samples from short-finned pilot whales (Globicephala macrorhynchus). We expanded upon a method for corticosteroid quantification by adding analytes and optimizing internal standard (IS) application.

We optimized a method for the quantification of seven steroid hormones using LC-MS/MS with a C18 column. We assessed the accuracy and precision of this updated C18 method and an existing biphenyl method for use with pilot whale blubber by conducting a spike recovery experiment and calculating percent recovery and relative standard deviation (RSD) for each analyte. To explore the potential for running this method with fewer matched ISs, we compared the performance of multiple ISs for each analyte.

All 11 adrenal and gonadal analytes showed good accuracy and precision in the spike recovery experiment, with recoveries between 82% and 110% and recovery RSDs below 10%. The C18 method detected all analytes at endogenous concentrations, except aldosterone. Although endogenous DHEA was detected, variability was high. IS comparisons showed 10 of 11 analytes could be calculated with comparable accuracy and precision using an IS substitute, but some substitutions significantly altered the analyte concentrations calculated.

The C18 method was not sensitive enough for endogenous aldosterone detection. DHEA, which has not been previously quantified in blubber, was detected in all samples, but with high variability at lower concentrations. The methods in this study provide reliable detection and quantification of the other nine hormones tested and can be used for assessments of adrenal and gonadal steroid hormones from whales. Laboratories can reduce costs through IS substitution but should consider how these substitutions affect results.

Immunoassays, which are used ubiquitously in clinical practice, are inherently vulnerable to distortions arising from endogenous immunoglobulins, particularly heterophilic antibodies. While many studies have explored interference in substances measured using chemiluminescence or electrochemiluminescence methods based on the double-antibody sandwich principle, there are limited data on interference in immunoturbidimetric assays, particularly for type IV collagen. This article presents the first report of a noteworthy increase in serum type IV collagen levels stemming from heterophilic antibody interference detected through an immunoturbidimetric assay. The present study investigated the mechanisms of this interference and the differences introduced by heterophilic antibodies between the two methodologies. Additionally, it outlines strategies for identifying and mitigating such interference, and discusses the principles, limitations, and considerations of each corrective approach. The objective is to raise awareness among clinical laboratory professionals concerning the potential interference of heterophilic antibodies in immunoturbidimetric assays. Increased awareness will aid in the prompt detection and correction of this issue, ensuring the provision of accurate and reliable laboratory data for informed clinical decision-making and the prevention of adverse medical outcomes.

Ovarian function suppression (OFS) has emerged as a crucial adjuvant therapy for premenopausal breast cancer patients. Some patients fail to achieve complete OFS with commonly used OFS drugs. The definition of incomplete OFS remains unclear, and large-scale data on its incidence are lacking. This review provides a comprehensive overview of the definition, occurrence, impact on therapeutic efficacy and corresponding treatment measures for incomplete OFS.

We searched PubMed, Embase and Cochrane Library databases in recent twenty years with keywords as "ovarian function escape", "incomplete OFS" and "estrogen breakthrough", and carried out a snowballing of references to important literature. Clinical literature of premenopausal breast cancer patients treated with OFS was screened. The patient characteristics, definition and incidence of incomplete OFS, prognosis, interventions and other information were extracted.

A total of 17 studies were included in the analysis, including RCTs, retrospective or prospective cohort studies and case reports. Literature indicates that the incidence of incomplete OFS is around 5-50 % when the estradiol (E2) threshold is set as 2.72 pg/mL, 10 pg/mL, 20 pg/mL, or 30 pg/mL. Young age, high body mass index (BMI), and no prior chemotherapy were the risk factors for incomplete OFS. The treatment of incomplete OFS included dose adjustments, alternative OFS drugs, or the adoption of other OFS measures.

The incomplete OFS rate decreased with the extension of treatment time. It is reasonable to monitor E2 levels to ensure successful OFS in the patients with high risk factors for incomplete OFS or with concurrent use of aromatase inhibitor (AI). Transient incomplete OFS seems to have no impact on prognosis, but sustained incomplete OFS needs personalized adjustment of treatment strategy to ensure complete OFS.

Our study evaluated the possible interference of Burosumab (human recombinant monoclonal antibody directed against N-terminal domain of FGF23) on the immunoassay of intact FGF23 (iFGF23) with the Liaison XL. The analytical method uses three different antibodies, one of which directed against the N-terminal portion of FGF23. The evaluation of the method accuracy involved the fully automated execution of a dilution test on EDTA plasma from 5 subjects who had not received any monoclonal antibody (mAb), 20 EDTA plasma from patients treated with Burosumab, and 2 EDTA plasma from subjects who had not received any mAb in witch an adequate volume of Burosumab had been added in vitro. One sample with specific diluent (LIAISON® FGF 23) with an adequate volume of Burosumab had been added in vitro. The dilution assay provided highly inaccurate iFGF23 results in samples with therapeutic concentrations of Burosumab and in samples with concentrations below the LoQ (6.5 pg/mL). The addition of Burosumab to the diluent did not produce any analytical interference. Dissociation of iFGF23 from the mAb-target complex in diluted sample could explain the loss of accuracy in the iFGF23 immunoassay using the Liaison XL analyzer. Burosumab could be an interferent in immunoassay procedures of iFGF23.

Assessment of hormone concentrations can be subjected to laboratory pitfalls. Macro-hormones are hormone-autoantibody complexes which are cleared slowly from circulation and cause a false elevation in hormones' concentrations. Macro-prolactin and macro-thyroid-stimulating hormone (TSH) are most frequently encountered while macro-follicle-stimulating hormone (FSH) has been rarely reported. We describe the case of a 30-year-old woman who had a gynaecological consultation due to failure in achieving pregnancy after 8 months of unprotected intercourse. She had regular menses, did not complain of climacteric symptoms and her medical history was unremarkable. Antral follicle count and anti-mullerian hormone concentrations were normal, and regular ovulation was documented. Unexpectedly, high early follicular phase FSH concentrations were confirmed on two occasions (57 and 51 IU/L), raising the suspicion of primary ovarian insufficiency. After excluding Turner's syndrome and autoimmune oophoritis, a laboratory artifact was hypothesized. Following polyethylene glycol precipitation, FSH levels dropped from 41.1 IU/L to 6.54 IU/L (recovery 16%) and the presence of macro-FSH was concluded. Laboratory interference can lead to misdiagnosis and unnecessary treatments. A laboratory artifact should be suspected when inconsistency exists between clinical presentation and laboratory results. Only five other cases of macro-FSH have been reported to date. Although macro-hormones generally have low biological activity and do not require treatment, the role of anti-FSH antibodies has been hypothesized in primary ovarian insufficiency and in vitro fertilization failure.

Hormone quantification is a cornerstone in the diagnostic workup of endocrine disorders, but it can be subjected to laboratory interferences which can lead to unnecessary investigations and inappropriate treatments. A laboratory artifact should be suspected when a discrepancy is observed between clinical presentation and laboratory results, when extremely unusual analyte concentrations are observed and when inconsistent results are obtained by different analytical methods. Macro-hormones are hormone-autoantibody complexes which are cleared slowly from circulation and cause a false elevation in hormone concentrations. Macro-prolactin and macro-TSH are most frequently encountered, while macro-FSH has been rarely reported. Macro-hormones can be detected by polyethylene glycol precipitation, gel filtration chromatography, or by using protein G or protein A columns. Although macro-hormones generally have low biological activity and do not require treatment, the role of anti-FSH antibodies has been hypothesized in primary ovarian insufficiency and in vitro fertilization failure.

Plasma levels of D-dimer are elevated in patients with thromboembolisms. Here we investigated the existence of interfering antibodies as a potential cause for elevated D-dimer levels.

A 42-year-old white Caucasian woman with a prior history of pulmonary embolism during her first pregnancy (treated with heparin therapy for 6 weeks postnatally) and hypothyroidism had a persistent elevated D-dimer without any clinical or ultrasound-based signs of thromboembolic conditions during her second pregnancy. We obtained informed consent and plasma was obtained from the patient. D-dimer levels were measured using two different assays. We also tested for the presence of rheumatoid factor, performed dilution series, and finally used an antibody depletion strategy. The two D-dimer assays performed similarly. Using our antibody depletion technique, we observed that ~ 1/3 of the increased plasma levels of D-dimer may be attributed to interfering antibodies.

Our results identify interfering antibodies as a potential contributor to an increased D-dimer in this patient. Our case highlights the potential of heterophilic interference for increased D-dimer and provides a procedure to determine this analytically.

Unexplained B12 hypervitaminosis (HB12) in asymptomatic patients leads to a cascade of medical consultations and diagnostic tests aimed at determining its etiology. The objective of this study was to assess the efficacy of the laboratory getting involved in the detection and elimination of immune complexes with vitamin B12 in clinical practice and its economic impact.

A retrospective longitudinal study was undertaken to assess the laboratory strategy of detecting B12 macrovitamin (macro-B12) in patients with HB12 >1,000 pg/mL. The clinical characteristics of patients with HB12 referred to Internal Medicine (IM) in the pre- and post-implantation period of the new strategy were compared. Additionally, the healthcare costs of one-year follow-up were estimated.

The prevalences of HB12 in the pre- and post-implantation period were 3.9 % and 3 %, respectively. Macro-B12 explained 25 % of the HB12 cases initially detected. A 41 % reduction was observed in the number of patients with HB12 after the implantation of the new strategy, thereby resulting in a cost reduction of 5,000 €.

The laboratory intervention for the detection of macro-B12 provides clear economic and clinical benefits in clinical practice.

Malaria has complex interactions with host physiology, including alterations in cortisol levels. Cortisol, a key hormone in the stress response, is known to be dysregulated in various infectious diseases. This systematic review and meta-analysis aimed to elucidate the relationship between Plasmodium infection and cortisol levels, shedding light on the intricate interplay between the parasite and the host's endocrine system. The methodological protocol for assessing cortisol levels in malaria patients was registered in PROSPERO (CRD42024496578), a widely recognized international prospective register of systematic reviews. This registration ensures transparency and minimizes the risk of bias in our research. A comprehensive search strategy was employed across major databases, including Embase, PubMed, Scopus, and Medline, to include studies that reported cortisol levels in infected patients. The qualitative synthesis was undertaken to synthesize the difference in cortisol levels between malaria-infected and uninfected individuals. The meta-analysis employed the random effects model in the quantitative synthesis to calculate the effect estimate. The review included a total of 20 studies, with a substantial number conducted in Africa, followed by Asia and South America. Most included studies (13/20, 65%) reported higher cortisol levels in infected patients than in uninfected patients. The meta-analysis confirmed significantly higher cortisol levels in infected patients compared to uninfected individuals (P < 0.0001, standardized mean difference (SMD): 1.354, 95% confidence interval: 0.913 to 1.795, I2: 88.3%, across 15 studies). Notably, the method for cortisol measurement and the type of blood sample used (serum or plasma) were significant moderators in the analysis, indicating that these factors may influence the observed relationship between Plasmodium infection and cortisol levels. The systematic review and meta-analysis confirmed that Plasmodium infection is associated with increased cortisol levels, highlighting the intricate relationship between the disease and the host stress response. These findings underscore the potential of cortisol as a supplementary biomarker for understanding the pathophysiological impact of malaria. By providing insights into the stress-related mechanisms of malaria, this comprehensive understanding can inform future research and potentially enhance disease management and treatment strategies, particularly in regions heavily burdened by malaria.

Quantifying small amounts of the 17-hydroxyprogesterone in various matrix is crucial for different purposes. In this study, a commercial polydimethylsiloxane stir bar was used to extract hormone from water and urine samples. Analysis was performed by high-performance liquid chromatography using a UV detector. The response surface methodology was used to optimize the desorption and extraction steps, with predicted optimal point relative errors of 1.25% and 6.40%, respectively. The optimized method was validated with a linear range of 1.21-1000.00 for aqueous and 2.43-2000.00 ng mL-1 for urine samples. The coefficient of determination was 0.9998 and 0.9967, and the detection limit of the proposed method was obtained to be 0.40 and 0.80 ng mL-1 for aqueous and urine samples, respectively. The recovery percentage and relative standard deviation within a day and between three days after the addition of three different concentration levels of the standard to the control sample were 87-103% and 0.4-3.6% for aqueous and 87.5-101% and 0.1-5.2% for urine samples, respectively. The results show that the proposed method can be appropriate and cost-effective for extracting and analyzing this hormone. In addition, using three different tools, the greenness of the proposed method was proven.

Measurements of aldosterone by mass spectrometry are more accurate and less prone to interferences than immunoassay measurements, and may produce a more accurate aldosterone:renin ratio (ARR) when screening for primary aldosteronism (PA).

Differences in diagnostic performance of the ARR using mass spectrometry vs immunoassay measurements of aldosterone were examined in 710 patients screened for PA. PA was confirmed in 153 patients and excluded in 451 others. Disease classifications were not achieved in 106 patients. Areas under receiver-operating characteristic curves (AUROC) and other measures were used to compare diagnostic performance.

Mass spectrometry-based measurements yielded lower plasma aldosterone concentrations than immunoassay measurements. For the ARR based on immunoassay measurements of aldosterone, AUROCs were slightly lower (P = 0.018) than those using mass spectrometry measurements (0.895 vs 0.906). The cutoff for the ARR to reach a sensitivity of 95% was 30 and 21.5 pmol/mU by respective immunoassay and mass spectrometry-based measurements, which corresponded to specificities of 57% for both. With data restricted to patients with unilateral PA, diagnostic sensitivities of 94% with specificities >81% could be achieved at cutoffs of 68 and 52 pmol/mU for respective immunoassay and mass spectrometry measurements.

Mass spectrometry-based measurements of aldosterone for the ARR provide no clear diagnostic advantage over immunoassay-based measurements. Both approaches offer limited diagnostic accuracy for the ARR as a screening test. One solution is to employ the higher cutoffs to triage patients likely to have unilateral PA for further tests and possible adrenalectomy, while using the lower cutoffs to identify others for targeted medical therapy.German Clinical Trials Register ID: DRKS00017084.

The diagnostic approach to hypopituitarism involves many disciplines. Clinical symptoms rarely are specific. Imaging techniques are helpful but cannot prove the specific functional defects. Therefore, the definitive diagnosis of pituitary insufficiency is largely based on laboratory tests. However, also laboratory methods come with inherent limitations, and it is essential for the clinician to know and recognize typical pitfalls. Most factors potentially impairing the quality of hormone measurements are introduced in the preanalytical phase, i.e. before the hormones are measured by the laboratory. For example, the timing of blood drawing with respect to circadian rhythm, stress, and medication can have an influence on hormone concentrations. During the actual analysis of the hormones, cross-reactions with molecules present in the sample presenting the same or similar epitopes than the intended analyte may affect immunoassays. Interference can also come from heterophilic or human anti-animal antibodies. Unexpected problems can also be due to popular nutritional supplements which interfere with the measurement procedures. An important example in this respect is the interference from biotin. It became only clinically visible when the use of this vitamin became popular among patients. The extreme serum concentrations reached when patients take it as a supplement can lead to incorrect measurements in immunoassays employing the biotin-streptavidin system. To some extent, hormone analyses using liquid chromatography mass spectrometry (LCMS) can overcome problems, although availability and cost-effectiveness of this method still imposes restrictions. In the post-analytical phase, appropriateness of reference intervals and cut-offs with respect to the specific analytical method used is of outmost importance. Furthermore, for interpretation, additional biological and pharmacological factors like BMI, age and concomitant diseases must be considered to avoid misinterpretation of the measured concentrations. It is important for the clinician and the laboratory to recognize when one or more laboratory values do not match the clinical picture. In an interdisciplinary approach, the search for the underlying cause should be initiated.

A small proportion of the patients with acromegaly present with apparently normal basal GH levels and suppressible GH levels despite increased IGF-1 levels, a pattern called micromegaly by some authors. Whether this pattern represents a distinct entity or is just an expression of acromegaly in its early stages is still a matter of debate. Nevertheless, these patients have some peculiar characteristics such as being more likely older and male, mostly harbour microadenomas or small macroadenomas, and have lower IGF-1 and postglucose GH levels. Even though, the frequency and severity of clinical signs and comorbidities are similar to those of patients with classic acromegaly. In conclusion, micromegaly seems to be a distinct clinical entity with a different biological behavior characterized by a low GH output.

A mass spectrometry (LC‒MS/MS)-based interlaboratory comparison study was performed for nine steroid analytes with five participating laboratories. The sample set contained 40 pooled samples of human serum generated from preanalyzed leftovers. To obtain a well-balanced distribution across reference intervals of each steroid, the leftovers first underwent a targeted mixing step.

All participants measured a sample set once using their own multianalyte protocols and calibrators. Four participants used in-house developed measurement platforms, including IVD-CE certified calibrators, which were used by three participants; the 5th lab used the whole LC‒MS kit from an IVD manufacturer. All labs reported results for 17-hydroxyprogesterone, androstenedione, cortisol, and testosterone, and four labs reported results for 11-deoxycortisol, corticosterone, cortisone, dehydroepiandrosterone sulfate (DHEAS), and progesterone.

Good or acceptable overall comparability was found in Bland‒Altman and Passing‒Bablok analyses. Mean bias against the overall mean remained less than ±10 % except for DHEAS, androstenedione, and progesterone at one site and for cortisol and corticosterone at two sites (max. -18.9 % for androstenedione). The main analytical problems unraveled by this study included a bias not previously identified in proficiency testing, operator errors, non-supported matrix types and higher inaccuracy and imprecision at lower ends of measuring intervals.

This study shows that intermethod comparison is essential for monitoring the validity of an assay and should serve as an example of how external quality assessment could work in addition to organized proficiency testing schemes.

Background/Objectives: Premenstrual dysphoric disorder (PMDD) is an understudied psychiatric condition affecting reproductive-age women who experience negative mood in the luteal phase of the menstrual cycle. Cognitive functions in PMDD are not well understood as patients have been tested in the luteal phase. This may confound study results due to noted emotional interferences, as well as the potential opposing effects of the sex hormones estradiol and progesterone. In the present study, we evaluated visuospatial function in the follicular phase in women with PMDD and healthy controls, and further examined the effect of estradiol as research into the hormonal mediation of visuospatial function in reproductive-age women has produced mixed results. Methods: To this end, we analyzed estradiol concentrations using the gold standard mass spectrometry. Serum samples were collected in the early follicular and mid/late follicular subphases when estradiol is low and high, respectively, while progesterone is low and steady. We assessed visuospatial function using the classic mental rotation task. Results: Women with PMDD had a higher mental rotation total score (t = 2.17; p < 0.05). The addition of six demographic, biological, and anthropomorphic variables in a hierarchical fashion accounted for 45.3% of the total variance in the final model with diagnosis remaining statistically significant (t = 4.36; p < 0.001). Estradiol did not mediate the group difference and was not significantly associated with visuospatial function. Conclusions: The present results provide support for new research directions into the potential biological mechanisms that underlie the pathophysiology of PMDD, represented as enhanced visuospatial ability in women with PMDD in the follicular phase. We review the theory that PMDD is a disorder of the enhanced excitation-to-inhibition ratio, with a focus on findings to date from brain imaging research.

Gestational diabetes is typically diagnosed in the late second or third trimester of pregnancy. It is one of the most common metabolic disorders among expectant mothers, with potential serious short- and long-term complications for both maternal and offspring health. C-peptide is secreted from pancreatic beta-cells into circulation in equimolar amounts with insulin. It is a useful biomarker to estimate the beta-cell function because it undergoes negligible hepatic clearance and consequently it has a longer half-life compared to insulin. Pregnancy induces increased insulin resistance due to physiological changes in hormonal and metabolic homeostasis. Inadequate compensation by islet beta-cells results in hyperglycemia. The standard oral glucose tolerance test at 24-28 wk of gestation sets the diagnosis. Accumulated evidence from prospective studies indicates a link between early pregnancy C-peptide levels and the risk of subsequent gestational diabetes. Elevated C-peptide levels and surrogate glycemic indices at the beginning of pregnancy could prompt appropriate strategies for secondary prevention.

Prolactin is a polypeptide hormone made of 199 amino acids; 50% of the amino acid chain forms helices, and the rest forms loops. This hormone is typically related to initiating and maintaining lactation, although it is also elevated in various pathological conditions. Serum prolactin levels of 2 to 18 ng ml-1 in men, up to 30 ng ml-1 in women, and 10 to 210 ng ml-1 in pregnant women are considered normal. Immunoassay techniques used for detection are susceptible to error in different clinical conditions. Surface-enhanced Raman spectroscopy (SERS) is a technique that allows for obtaining the protein spectrum in a simple, fast, and reproducible manner. Nonetheless, proper characterization of human prolactin's Raman/SERS spectrum at different concentrations has so far not been deeply discussed. This study aims to characterize the Raman spectrum of human prolactin at physiological concentrations using silver nanoparticles (AgNPs) as the SERS substrate. The Raman spectrum of prolactin at 20 ng ul-1 was acquired. Quasi-spherical AgNPs were obtained using chemical synthesis. For SERS characterization, decreasing dilutions of the protein were made by adding deionized water and then a 1 : 1 volume of the AgNPs colloid. For each mixture, the Raman spectrum was determined. The spectrum of prolactin by SERS was obtained with a concentration of up to 0.1 ng ml-1. It showed characteristic bands corresponding to the side chains of aromatic amino acids in the protein's primary structure and the alpha helices of the secondary structure of prolactin. In conclusion, using quasi-spherical silver nanoparticles as the SERS substrate, the Raman spectrum of human prolactin at physiological concentration was determined.

The gonadotropin-releasing hormone receptor variant GNRHR p.Q106R (rs104893836) in homozygosity, compound heterozygosity, or single heterozygosity is often reported as the causative variant in idiopathic hypogonadotropic hypogonadism (IHH) patients with GnRH deficiency. Genotyping of a Maltese newborn cord-blood collection yielded a minor allele frequency (MAF) 10 times higher (MAF = 0.029; n = 493) than that of the global population (MAF = 0.003).

To determine whether GNRHR p.Q106R in heterozygosity influences profiles of endogenous hormones belonging to the hypothalamic-pituitary axis and the onset of puberty and fertility in adult men (n = 739) and women (n = 239).

Analysis of questionnaire data relating to puberty and fertility, genotyping of the GNRHR p.Q106R variant, and hormone profiling of a highly phenotyped Maltese adult cohort from the Maltese Acute Myocardial Infarction Study.

Out of 978 adults, 43 GNRHR p.Q106R heterozygotes (26 men and 17 women) were identified. Hormone levels and fertility for all heterozygotes are within normal parameters except for TSH, which was lower in men 50 years or older.

Hormone data and baseline fertility characteristics of GNRHR p.Q106R heterozygotes are comparable to those of homozygous wild-type individuals who have no reproductive problems. The heterozygous genotype alone does not impair the levels of investigated gonadotropins and sex steroid hormones or affect fertility. GNRHR p.Q106R heterozygotes who exhibit IHH characteristics must have at least another variant, probably in a different IHH gene, that drives pathogenicity. We also conclude that GNRHR p.Q106R is likely a founder variant due to its overrepresentation and prevalence in the island population of Malta.

Reproductive biomarkers are important regulators in women, especially during pregnancy and childbirth. Because of their essential role in women's health, the discovery and quantification of reproductive biomarkers is of great clinical importance. Nowadays, there are many detection strategies to detect these biomarkers, including VEGF, human chorionic gonadotropin (hCG), etc. Consider the limitations and problems of conventional diagnostic methods, new methods are being developed, one of the most important being methods based on nanotechnology. This review includes a review of methods for diagnosing reproductive biomarkers, ranging from mainstream to nanotechnology-based methods. The bulk of this article is an in-depth introduction to the latest advances in biosensor and nanosensor research for the detection and quantitative identification of reproductive biomarkers.

Polycystic ovary syndrome (PCOS) is a prevalent hormonal disorder that affects women of reproductive age. It is characterized by abnormal production of androgens, typically present in small quantities in females. This study aimed to investigate the therapeutic potential of Irosustat (STX64), STX140, and compound 1G as new drug candidates for the treatment of letrozole-induced PCOS in female Wistar rats. 36 rats were divided into six groups of equal size. PCOS was induced in all groups, except the normal control group, by administering letrozole orally (1 mg/kg/day for 35 days). The onset of abnormal estrous cycle was confirmed by examining daily vaginal smears under a microscope. Subsequently, each rat group was assigned to a different treatment regimen, including one control group, one letrozole group, one metformin group (500 mg/kg/day) as a reference drug, and the other groups received a different drug candidate orally for 30 days. After treatment, blood collection was performed for biochemical measurements and determination of oxidative stress markers. The rats were dissected to separate ovaries and uterus for morphological, histological, and western blotting studies. Treatment with the drug candidates improved the ovaries and uterus weight measurements compared to the untreated PCOS group. The three tested drug candidates demonstrated promising improvements in lipid profile, blood glucose level, testosterone, progesterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and estradiol levels. In addition, western blotting confirmed their promising effects on Akt, mTOR, and AMPK-α pathways. This study led to the discovery of three promising drug candidates for the management of PCOS as alternatives to metformin.

Calcitonin (CT) is a diagnostic and follow-up marker of medullary thyroid carcinoma. Heterophile antibodies (HAbs) may interfere during immunometric assay measurements and result in falsely high CT levels and different markers. A 50-year-old female patient was referred to our institution for elevated CT levels (3,199 pg/mL [0-11,5]). Physical examination and thyroid ultrasonography show no thyroid nodules. Because of the discrepancy between the clinical picture and the laboratory results, various markers and hormones were examined to determine whether there was any interference in the immunometric assay. Thyroglobulin (Tg) and Adrenocorticotropic hormone (ACTH) levels were also found inaccurately elevated. After precipitation with polyethylene glycol, CT, Tg, and ACTH levels markedly decreased, showing macro-aggregates. Also, serial dilutions showed non-linearity in plasma concentrations. Additionally, CT samples were pretreated with a heterophilic blocking tube before measuring, and the CT level decreased to < 0.1 pg/mL, suggesting a HAb presence. Immunoassay interference should be considered when conflicting laboratory data are observed. This may help reduce the amount of unnecessary laboratory and imaging studies and prevent patients from complex diagnostic procedures.

Method-related variations in the measurement of hormones and the reference intervals used in the clinical laboratory can have a significant, but often under-appreciated, impact on the diagnosis and management of endocrine disorders. This variation in laboratory practice has the potential to lead to an errant approach to patient care and thus could cause harm. It may also be the source of confusion or result in excessive or inadequate investigation. It is important that laboratory professionals and clinicians know about these impacts, their sources, and how to detect and mitigate them when they do arise. In this review article, we describe the historical and scientific context from which inconsistency in the clinical laboratory arises. Examples from the published literature of the impact of the method, reference interval, and clinical decision threshold-related discordances on the assessment and monitoring of various endocrine disorders are discussed to illustrate the sources, causes, and effects of this variability. Its potential impact on the evaluation of growth hormone deficiency and excess, thyroid and parathyroid disorders, hyperandrogenism, hypogonadism, glucocorticoid excess and deficiency, and diabetes mellitus is elaborated. Strategies for assessment and mitigation of the discordance are discussed. The clinical laboratory has a responsibility to recognise and address these issues, and although a lot has been accomplished in this area already, there remains more to be done.

Early evidence suggests that hormonal contraceptive (HC) use alters psychological functioning and competitive behavior. Yet, there is limited data on endocrine models for explaining how HC use affects these outcomes. In this pre-registered and open-data study, we test if HC users and naturally cycling (NC) females in their low (mid-follicular) and high (mid-luteal) progesterone phase differ in competitive persistence and whether progesterone and cortisol reactivity mediate of this effect. HC users (N = 73) in the active hormone-exposure phase and NC participants in the mid-follicular (N = 69) or mid-luteal (N = 72) phase completed two behavioral measures of competitive persistence, holding up a weight for time followed by attempting to solve an unsolvable anagram. Participants also completed measures of handgrip strength and self-reported competitiveness as well as gave saliva samples before and after the tasks for hormone assay. Results showed that NC-follicular group had greater competitive persistence in the weight-holding task compared to both NC-luteal (d = 0.38) and HC use (d = 0.43) groups independent of physical strength and self-reported competitiveness covariates. Although anagram task performance showed similar trends for group differences, analyses for this task were inconclusive. Baseline progesterone did not mediate the effect of cycle phase group on competitive persistence. HC users showed relatively blunted cortisol and progesterone reactivity, and this effect partially mediated the difference in competitive persistence between HC users and the NC-follicular group. In sum, results suggest that HC use could downregulate competitive behavior at least partly by dampening cortisol-progesterone reactivity. These findings offer a new endocrine model for understanding HC use and cycle phase effects on motivational and energetic outcomes required for optimal performance in competitive contexts.

Fulvestrant resembles estradiol in its structure. Reports have been published concerning fulvestrant measured as estradiol by the immunoassays. This may induce falsely elevated estradiol results and wrongly impact medical decisions. Our aim was to confirm the interference of fulvestrant on estradiol concentration and test a method to identify the false results.

Four serum samples with low estradiol levels were spiked with fulvestrant at various concentrations. Estradiol was then measured directly on serum (Dir), after a 1:5 dilution (Dil), and a ratio Dil/Dir was estimated. On the second part of the study, estradiol results (Dir, Dil and ratio Dil/Dir) from 14 women treated with fulvestrant were analysed, as well as from 14 patients not under this treatment.

The addition of exogenous fulvestrant to the serum samples induced a gradual rise on estradiol concentration with a mean ratio for the Dil/Dir samples of 2.1 ± 0.4 (range 1.7-2.9). Patients on fulvestrant treatment experienced a mean ratio for the Dil/Dir estradiol sample of 2.4 ± 0.4 (range 1.6-3.0). In the control group, a mean estradiol ratio Dil/Dir of 1.1 ± 0.1 was observed (range 0.8-1.3). No correlation between the number of days after fulvestrant injection and estradiol result (r = 0.531) was observed.

Our study confirmed the interference of fulvestrant in the estradiol measurement by immunoassay. When fulvestrant was present, the estradiol ratio for Dil/Dir sample was about 2. In the control group, the ratio was around 1. The estradiol Dil/Dir ratio is a simple tool which can be used to identify fulvestrant false immunoassay estradiol results.

An immunoassay is an analytical test method in which analyte quantitation is based on signal responses generated as a consequence of an antibody-antigen interaction. They are the method of choice for the measurement of a large panel of diagnostic markers. Not only are they fully automated, allowing for a short turnaround time and high throughput, but offer high sensitivity and specificity with low limits of detection for a wide range of analytes. Many immunoassay manufacturers exploit the extremely high affinity of biotin for streptavidin in their assay design architectures as a means to immobilize and detect analytes of interest. The biotin-(strept)avidin system is, however, vulnerable to interference with high levels of supplemental biotin that may cause elevated or suppressed test results. Since this system is heavily applied in clinical diagnostics, biotin interference has become a serious concern, prompting the FDA to issue a safety report alerting healthcare workers and the public about the potential harm of ingesting high levels of supplemental biotin contributing toward erroneous diagnostic test results. This review includes a general background and historical prospective of immunoassays with a focus on the biotin-streptavidin system, interferences within the system, and what mitigations are applied to minimize false diagnostic results.

The Dengue virus complex (DENV), formed by four serotypes, constitutes the most important arbovirus affecting humans. The structural domain III of their envelope protein (DIII) elicits strongly neutralizing serotype-specific antibodies. Contrasting results have been obtained regarding their role in the serum neutralizing activity of infected patients. We used a DENV immune serum from a secondary infection to examine the impact of characterizing the anti-DIII antibody response after affinity purification with recombinant DIII proteins to eliminate potential interferences from the interactions with human plasma proteins and other anti-DENV antibodies. Total anti-DENV IgG repertoire and anti-DIIIE antibodies were compared in functionality. In early convalescence, reactivity of anti-DIII antibodies is serotype specific and exhibits the strongest reactivity with infecting serotypes. Purification of anti-DIII antibodies emphasizes the reactivity profile as compared to total IgG fraction and serum. Serotype-specificity of the virus neutralization activity correlated with the apparent kD of the binding to recombinant DIIIs.

Seminal plasma cytokines are associated with fertility and reproductive health, but progressing their clinical utility is hampered by absence of reference data on concentration ranges of relevant cytokines in healthy men. We employed a systematic approach to assemble current evidence on the concentrations of immune regulatory cytokines present in seminal plasma (SP) of normozoospermic and/or fertile men and evaluated the impact of different platform methodologies for cytokine quantification.

A systematic literature search was performed utilising PubMed, Web of Science and Scopus. Databases were searched from inception until 30th June 2022 inclusive, using combinations of keywords pertaining to seminal fluid and cytokines, and was restricted to human participants. Original data with values reported as concentration of specific cytokines in SP of men clearly defined as fertile or normozoospermic were extracted from studies written in English.

A total of 3769 publications were initially identified, of which 118 fulfilled the eligibility criteria for inclusion. A total of 51 individual cytokines are detectable in SP of healthy men. The number of studies reporting on each cytokine range from 1 to >20. The reported concentrations for many cytokines linked with fertility status, including IL6, CXCL8/IL8, and TNFA, are highly variable between published studies. This is associated with the different immunoassay methodologies utilised and may be exacerbated by a lack of validation of assays to ensure suitability for SP assessment. Due to the large variation between studies, accurate reference ranges for healthy men cannot be determined from the published data.

The concentrations of cytokines and chemokines detected in SP is inconsistent and highly variable between studies and cohorts, limiting current capacity to define reference ranges for cytokine concentrations in fertile men. The lack of standardisation in methods used to process and store SP, and variation in platforms used to evaluate cytokine abundance, are factors contributing to the observed heterogeneity. To progress the clinical utility of SP cytokine analysis will require standardisation and validation of methodologies so that reference ranges for healthy fertile men can be defined.

The clinical consequences of primary hypothyroidism include cardiovascular morbidity, increased mortality, and poor quality of life; therefore guidelines endorsed by several Scientific Societies recommend measuring circulating thyroid-stimulating hormone (TSH) in patients at risk. The assessment of serum TSH levels is also deemed to be the most robust and accurate biomarker during the management of replacement therapy in patients with a previous diagnosis of primary hypothyroidism. In line with a reflex TSH laboratory strategy, free thyroxine is measured only if the TSH falls outside specific cutoffs, in order to streamline investigations and save unjustified costs. This serum TSH-based approach to both diagnosis and monitoring has been widely accepted by several national and local health services; nevertheless, false-negative or -positive testing may occur, leading to inappropriate management or treatment. This review aims to describe several infrequent causes of increased circulating TSH, including analytical interferences, resistance to TSH, consumptive hypothyroidism, and refractoriness to levothyroxine replacement treatment. We propose a clinical flowchart to aid correct recognition of these various conditions, which represent important potential pitfalls in the diagnosis and treatment of primary hypothyroidism.

Lipemia is one of common endogenous interferences that can compromises sample quality and potentially influence results of various laboratory methods. Determination of the lipemic index or triglyceride concentrations are used to define the degree of lipemia. This study was aimed to establish lipemic index (LI) and triglyceride thresholds above where significant interference exists for 31 immunoassay analytes measured on Roche Cobas 6000.

The study was carried out following CLSI C56-A and EP07-ED3:2018 guidelines using sample pools spiked with increasing concentrations of lipid emulsion solution, reaching 70 mmol/L. To define the LI and triglyceride thresholds, the bias from concentration in the native sample was calculated at different lipemia degree and compared with allowable error limits based on biological variation or state-of-the-art technology.

No lipemia interference was observed for 27 out of 31 analytes even at the highest concentrations of lipid emulsion (LI ranging from 1737 to 2086 mg/dL, triglyceride concentration 60.34-73.99 mmol/L). However, progesterone, 25-OH vitamin D, testosterone, and estradiol were negatively affected by lipemia at 217 mg/dL (9.58 mmol/L), 222 mg/dL (10.66 mmol/L), 478 mg/dL (18.81 mmol/L), and 941 mg/dL (35.82 mmol/L) of the LI (triglyceride concentration), respectively.

Most immunoassays evaluated in this study were found to be robust to lipemia interference. By using these thresholds, laboratories can report the immunoassay results from analyzing a lipemic patient sample in many cases.

Measurement of hormones is important for the diagnosis and management of endocrine diseases. The thyroid hormones thyroxine (T4) and triiodothyronine (T3) are among the most commonly measured hormones in clinical laboratories, and it is the concentration of free (not bound to proteins) thyroid hormones that is clinically most relevant. Free thyroid hormones are commonly measured using automated immunoassays, however, these are known to produce erroneous results due to interferences for some patients. Measurement of free thyroid hormones using equilibrium dialysis or ultrafiltration combined with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is considered a more accurate and robust method for free thyroid hormone analysis and overcomes many of the limitations of immunoassays. However, LC-MS/MS-based methods are often considered too technically difficult and not amendable to high throughput by clinical chemists and are not offered by many clinical laboratories. This mini-review aims to make it easier for clinical laboratories to implement LC-MS/MS-based measurement of free thyroid hormones. It describes the medical rationale for measuring free thyroid hormones, the benefits of LC-MS/MS-based methods with respect to interferences affecting immunoassay-based methods and physical separation methods. This mini-review highlights important parameters for ultrafiltration and equilibrium dialysis to obtain physiologically relevant free thyroid hormone concentrations and focuses on methods and devices used in clinical chemistry.

Thyroid-stimulating hormone (TSH) and thyroid hormone levels are standard parameters in blood analysis. However, the immunoassays employed may lead to false-positive or false-negative results when the sample contains certain materials that interfere with the assay. Macro-TSH, a complex of TSH with immunoglobulin or albumin, may cause apparently increased TSH concentrations. TSH is produced in the pars tuberalis (PT) of the pituitary gland and by thyrotrophs of the pars distalis (PD). It was found that variable glycosylation can render the molecule more strongly bound to antibodies or albumin in the blood, leading to the hypothesis that macro-TSH consists mainly of PT-TSH. Although less known than PD-TSH, PT-TSH plays an important role in the central regulation of thyroid metabolism. The present review summarizes the physiological function of human PT-TSH and its role in macro-TSH formation. The prevalence of macro-hyperthyrotropinemia, the structure of PT-TSH and macro-TSH, problems in the measurement of TSH, and the action of PT-TSH in animals with seasonal breeding are discussed. Despite the absence of a specific function of macro-TSH in the organism, the identification of macro-TSH is important for avoiding unnecessary treatment based on a falsified readout of increased TSH concentrations as numerous individual case reports describe.

In the last decade, the combination of the widespread use of streptavidin-biotin technology and biotin-containing supplements (BCS) in the daily clinical practice, have led to numerous reports of erroneous hormone immunoassay results. However, there are no studies assessing the clinical and biochemical significance of that phenomenon, when treating patients with hypothyroidism. Therefore, a prospective study was designed to investigate the potential alterations in the measurement of thyroid hormone concentrations and clinical consequences in patients with hypothyroidism using low -dose BCS containing less than 300 μg/day.

Fifty-seven patients on thyroxine supplementation, as a result of hypothyroidism and concurrent use of BCS at a dose <300μg/day for 10 to 60 days were prospectively evaluated. Namely, TSH and free T4 (FT4) concentration measurements were performed, during BC supplementation and 10 days post BCS discontinuation and compared to 31 age-matched patients with supplemented hypothyroidism and without BCS.

A statistically significant increase in TSH and decline in FT4 concentrations was observed after BCS discontinuation. However, on clinical grounds, these modifications were minor and led to medication dose adjustment in only 2/57 patients (3.51%) in whom TSH was notably decreased after supplement discontinuation.

Our study suggests that changes in thyroid hormones profiling, due to supplements containing low dose biotin, are of minimal clinical relevance and in most cases don't occult the need to adjust the thyroxine replacement dose in patients with hypothyroidism. Larger, well-designed trials are required to further evaluate this phenomenon.

Sex hormone concentrations, particularly testosterone, are primary determinants of sex-based differences in athletic and sports performance, and this relationship may inform fair competition and participation for athletes. This article describes the sex-based dichotomy in testosterone and the implications for sex-based differences in individual sports performance, including factors that relate to athletic performance for transgender individuals, and areas of future investigation.

The objective of this study was to collect information on human chorionic gonadotrophin (hCG) laboratory testing and reporting in women with gestational trophoblastic disease (GTD), to assess the associated challenges, and to offer perspectives on hCG testing harmonisation.

Information was collected from laboratories by electronic survey (SurveyMonkey) using a questionnaire designed by members of the European Organisation for the Treatment of Trophoblastic Disease (EOTTD) hCG working party.

The questionnaire was distributed by the EOTTD board to member laboratories and their associated scientists who work within the GTD field.

The questionnaire was distributed and accessed via an online platform.

The questionnaire consisted of 5 main sections. These included methods used for hCG testing, quality procedures, reporting of results, laboratory operational aspects, and non-GTD testing capability. In addition to reporting these survey results, examples of case scenarios which illustrate the difficulties faced by laboratories providing hCG measurement for GTD patient management were described. The benefits and challenges of using centralised versus non-centralised hCG testing were discussed alongside the utilisation of regression curves for management of GTD patients.

Information from the survey was collated and presented for each section and showed huge variability in responses across laboratories even for those using the same hCG testing platforms. An educational example was presented, highlighting the consequence of using inappropriate hCG assays on clinical patient management (Educational Example A), along with an example of biotin interference (Educational Example B) and an example of high-dose hook effect (Educational Example C), demonstrating the importance of knowing the limitations of hCG tests. The merits of centralised versus non-centralised hCG testing and use of hCG regression curves to aid patient management were discussed.

To ensure the survey was completed by laboratories providing hCG testing for GTD management, the questionnaire was distributed by the EOTTD board. It was assumed the EOTTD board held the correct laboratory contact, and that the questionnaire was completed by a scientist with in-depth knowledge of laboratory procedures.

The hCG survey highlighted a lack of harmonisation of hCG testing across laboratories. Healthcare professionals involved in the management of women with GTD should be aware of this limitation. Further work is needed to ensure an appropriate, quality-assured laboratory service is available for hCG monitoring in women with GTD.

Biochemical confirmation of a diagnosis of hypercortisolism (Cushing syndrome) is vital to direct further investigations, especially given the overlap with non-autonomous conditions, such as pseudo-Cushing, and the morbidity associated with missed diagnoses. A limited narrative review was performed focusing on the laboratory perspective of the pitfalls of making a biochemical diagnosis of hypercortisolism in those presenting with presumed Cushing syndrome. Although analytically less specific, immunoassays remain cheap, quick, and reliable in most situations. Understanding cortisol metabolism can help with patient preparation, specimen selection (e.g., consideration of urine or saliva for those with possible elevations of cortisol binding globulin concentration), and method selection (e.g., mass spectrometry if there is a high risk of abnormal metabolites). Although more specific methods may be less sensitive, this can be managed. The reduction in cost and increasing ease of use makes techniques such as urine steroid profiles and salivary cortisone of interest in future pathway development. In conclusion, the limitations of current assays, particularly if well understood, do not impede diagnosis in most cases. However, in complex or borderline cases, there are other techniques to consider to aid in the confirmation of hypercortisolism.

We present an enzyme-like functional polymer that recognizes nonelectroactive targets and catalyzes their redox reactions for simple, selective steroid metabolite detection. Measuring steroid metabolites, such as cortisol, has been widely adopted to diagnose stress and chronic diseases. Conventional detection method based on competitive immunoassay requires time-consuming labeling processes for signal transduction and unstable biological receptors for biorecognition yet with limited selectivity. Inspired by natural enzymes' target specificity and catalytic nature, we report an enzyme-mimic using electrocatalytic molecularly imprinted polymers (EC-MIP) to achieve label-free, external redox reagent-free, sensitive, and selective electrochemical detection of cortisol. The EC-MIP sensor contains molecularly imprinted cavities for specific cortisol binding and embedded copper phthalocyanine tetrasulfonate (CuPcTS) for electrocatalytic reduction of the ketones on the captured cortisol into alcohols. The direct sensing approach resolves the intrinsic limitations of conventional MIP-based sensors, most notably the use of external redox probes and weak sensing signals. The sensor exhibited a detection limit of 181 pM with significantly enhanced selectivity using a differential sensing mechanism. The new enzyme-like sensor can be modified to detect other targets, offering a simple, robust approach to future health monitoring technologies.

Civil aviation flight crew and civil aviation air traffic controllers are prone to circadian rhythm abnormalities, which can lead to a slew of other maladies. It could endanger people's health and provide a serious threat to the safety of civil aviation flights if it is not appropriately evaluated and addressed. Early detection of rhythm irregularities and prompt treatment for particular populations that are vulnerable to rhythm disorders are crucial for enhancing civil aviation safety. In general, monitoring of the classical circadian rhythm biomarkers (melatonin or cortisol) in plasma or saliva is an effective way to evaluate the rhythm status. Due to the challenging sample procedure and the trauma of plasma, urine sample testing has received an increasing amount of attention. While, urine circadian rhythm biomarkers have seldom been examined, and the relationship between urinary steroid hormones and melatonin is still poorly understood. In most cases, hormones are determined by immunoassays respectively, mainly enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA). There are also reports describing the liquid chromatography with tandem mass spectrometry (LC-MS/MS) technique as a method of melatonin or few steroid hormones quantification, however, the simultaneous detection of multiple rhythmic hormones in human urine is rarely reported. For the quantification of the rhythmic hormones in human urine, an accurate approach using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was devised in this work. Nine endogenous hormones (melatonin, 6-hydroxymelatonin, 6-sulfatoxymelatonin, cortisol, corticosterone, cortisone, testosterone, epitestosterone and androsterone), in human overnight urine, were quantified after solid phase extraction (SPE). A reverse phase HSS C18 column was used for chromatographic separation with a 9-minute gradient elution and deuterated analogues of each analyte were applied as internal standards. This method was successfully applied to the analysis of 596 overnight urine samples (23:00-9:00) collected from 84 air traffic controllers in the Beijing area during shift work. This study's findings showed a clear correlation not only between melatonin and its metabolites; cortisol-related metabolites, but also between melatonin metabolites and endogenous metabolites upstream and downstream of cortisol, implying that these two categories of hormones can be used as potential biological rhythm indicators to provide circadian rhythm data support for future studies on circadian rhythm disorders.

Thyroid dysfunctions are among the most common endocrine disorders and accurate biochemical testing is needed to confirm or rule out a diagnosis. Notably, true hyperthyroidism and hypothyroidism in the setting of a normal thyroid-stimulating hormone level are highly unlikely, making the assessment of free thyroxine (FT4) inappropriate in most new cases. However, FT4 measurement is integral in both the diagnosis and management of relevant central dysfunctions (central hypothyroidism and central hyperthyroidism) as well as for monitoring therapy in hyperthyroid patients treated with anti-thyroid drugs or radioiodine. In such settings, accurate FT4 quantification is required. Global standardization will improve the comparability of the results across laboratories and allow the development of common clinical decision limits in evidence-based guidelines. The International Federation of Clinical Chemistry and Laboratory Medicine Committee for Standardization of Thyroid Function Tests has undertaken FT4 immunoassay method comparison and recalibration studies and developed a reference measurement procedure that is currently being validated. However, technical and implementation challenges, including the establishment of different clinical decision limits for distinct patient groups, still remain. Accordingly, different assays and reference values cannot be interchanged. Two-way communication between the laboratory and clinical specialists is pivotal to properly select a reliable FT4 assay, establish reference intervals, investigate discordant results, and monitor the analytical and clinical performance of the method over time.

Bacteria, viruses, and parasites are harmful microorganisms that cause infectious diseases. Early detection of diseases is critical to prevent disease transmission and provide epidemic preparedness, as these can cause widespread deaths and public health crises, particularly in resource-limited countries. Lateral flow assay (LFA) systems are simple-to-use, disposable, inexpensive diagnostic devices to test biomarkers in blood and urine samples. Thus, LFA has recently received significant attention, especially during the pandemic. Here, first of all, the design principles and working mechanisms of existing LFA methods are examined. Then, current LFA implementation strategies are presented for communicable disease diagnoses, including COVID-19, zika and dengue, HIV, hepatitis, influenza, malaria, and other pathogens. Furthermore, this review focuses on an overview of current problems and accessible solutions in detecting infectious agents and diseases by LFA, focusing on increasing sensitivity with various detection methods. In addition, future trends in LFA-based diagnostics are envisioned.

For over 50 years, immunoassays have been extensively used to quantitate hormones in blood, other fluids and tissues. Each assay has its own sensitivity, specificity and other analytical components. Despite the differences between commercial products, these assays provide important clinical information about hormone levels in patients. However, inaccurate results can occur because of technical issues, as well as patient-specific factors that can interfere with immunoassay hormone measurements. The latter include excessive normal blood or serum components, the presence of cross-reacting substances, extremely high levels of hormones leading to the high-dose hook effect, and interference from a variety of endogenous factors such as human antibodies that interact with the assay components or high levels of biotin in the serum from exogenous ingestion. This article briefly reviews the sources and recognition of endogenous interference, and describes methods to determine the correct serum hormone concentration.

Anti-Müllerian hormone (AMH) is one of the most reliable markers of ovarian reserve. Automated AMH assays are widely used in clinical laboratories, but reference intervals for the Elecsys AMH assay for Asian populations have not yet been determined. We aimed to determine reference intervals in healthy Korean women.

The study included 1,450 women aged 19 to 54 years who participated in the Korea National Health and Nutrition Examination Survey between 2013 and 2016. The study participants were divided into seven 5-year age groups. AMH and progesterone concentrations were measured using Roche Elecsys assays, and bone morphogenetic protein-15 (BMP15) was genotyped for the detection of major variants. Age group-specific reference intervals for AMH were established as recommended by the CLSI EP28-A3c guidelines.

The mean age was 37.4 years. AMH concentrations decreased with increasing age, especially after 40 years, with the median AMH decreasing from 30.9 pmol/L in participants of 19-24 years to 0.071 pmol/L in participants of 50-54 years. The mid-95 percentile AMH reference intervals decreased from 7.93-81.21 pmol/L in participants of 19-24 years to 0.07-3.86 pmol/L in participants of 50-54 years. Disease-associated BMP15 variants were not detected.

We determined Elecsys AMH assay reference intervals in healthy Korean women. The results may provide basic information for the interpretation of AMH concentrations and assessment of ovarian reserve in Korean women.

Unconjugated estriol (uE3) is an important biomarker in second trimester prenatal screening. Previous studies from our laboratory identified rare interference in the Beckman uE3 assay due to anti-ALP antibodies, which could be mitigated with a scavenger or heat-inactivated ALP (hALP). In the current study, 160 de-identified patient samples previously submitted for the Quad screen with low uE3 multiples of the median (MoM ≤0.50) were investigated for potential interference.

A reagent pack spiking strategy with hALP was employed to understand if the interference could be identified and mitigated in a scalable manner. The 160 samples were measured using uE3 lot #920861 previously known to be subject to interference, lot #920861 spiked with hALP, and the vendor reformulated lot #922579. Samples were suspected to have interference if the percent difference in uE3 measurements was >50%. Pseudo-risks were calculated using a test patient environment to understand the screening impact due to the change in uE3 result.

Seventeen of the 160 samples had uE3 results that were >50% different between the hALP spiked and non-spiked reagent pack. Both original lot #920861 with hALP and reformulated lot #922579 identified the same 17 patients as having interference in lot #920861. Analysis of screening risks using a test patient environment showed that assay interference could result in false positives for one trisomy 21 and three trisomy 18 post-test risk calculations.

Our experiment of reagent pack spiking with hALP produced similar uE3 results to a reformulated reagent designed to address potential interference, demonstrating that this is a feasible strategy to screen for interference in a scalable manner. The vendor-provided reformulation addressed anti-ALP interference and improved the performance of the screen.

Accurate measurement of cortisol is critical in adrenal insufficiency as it reduces the risk associated with misdiagnosis and supports the optimization of stress dose. Comprehensive assays have been developed to determine the levels of bioactive free cortisol and their clinical and analytical efficacies have been extensively discussed because the level of total cortisol is affected by changes in the structure or circulating levels of corticoid-binding globulin and albumin, which are the main reservoirs of cortisol in the human body. Antibody-based immunoassays are routinely used in clinical laboratories; however, the lack of molecular specificity in cortisol assessment limits their applicability to characterize adrenocortical function. Improved specificity and sensitivity can be achieved by mass spectrometry coupled with chromatographic separation methods, which is a cutting-edge technology to measure individual as well as a panel of steroids in a single analytical run. The purpose of this review is to introduce recent advances in free cortisol measurement from the perspectives of clinical specimens and issues associated with prospective analytical technologies.