Pre-analytical stability and physiological fluctuations affect plasma steroid hormone outcomes: A real-world study.

Since steroids are crucial for diagnosing endocrine disorders, the lack of research on factors that affect hormone levels makes interpreting the results difficult. Our study aims to assess the stability of the pre-analytical procedure and the impact of hormonal physiological fluctuations using real-world data. The datasets were created using 12,418 records from individuals whose steroid hormone measurements were taken in our laboratory between September 2019 and March 2024. 22 steroid hormones in plasma by a well-validated liquid chromatography tandem mass spectrometry method were measured. After normalization transformation, outlier removal, and z-score normalization, generalized additive models were constructed to evaluate preanalytic stability and age, sex, and sample time-dependent hormonal fluctuations. Most hormones exhibit significant variability with age, particularly steroid hormone precursors, sex hormones, and certain corticosteroids such as aldosterone. 18-hydroxycortisol, 18-oxocortisol. Sex hormones varied between males and females. Levels of certain hormones, including cortisol, cortisone, 11-deoxycortisol, 18-hydroxycortisol, 18-oxocortisol, corticosterone, aldosterone, estrone, testosterone, dihydrotestosterone, dehydroepiandrosterone sulfate, 11-ketotestosterone, and 11-hydroxytestosterone, fluctuated with sampling time. Moreover, levels of pregnenolone and progesterone decreased within 1 hour of sampling, with pregnenolone becoming unstable with storage time at 4 degrees after centrifugation, while other hormone levels remained relatively stable for a short period of time without or after centrifugation of the sample. This is the first instance real-world data has been used to assess the pre-analytic stability of plasma hormones and to evaluate the impact of physiological factors on steroid hormones.

Liquid chromatography-tandem mass spectrometry in clinical laboratory protein measurement.

Proteins are essential components of human cells and tissues, and they are commonly measured in clinical laboratories using immunoassays. However, these assays have certain limitations, such as non-specificity binding, insufficient selectivity, and interference of antibodies. More sensitive, accurate, and efficient technology is required to overcome these limitations. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful analytical tool that provides high sensitivity and specificity, making it superior to traditional methods such as biochemical methods and immunoassays. While LC-MS/MS has been increasingly used for detecting small molecular analytes and steroid hormones in clinical practice recently, its application for protein or peptide analysis is still in its early stages. Established methods for quantifying proteins and peptides by LC-MS/MS are mainly focused on scientific research, and only a few proteins and peptides can be or have the potential to be detected and applied in clinical practice. Therefore, this article aims to review the clinical applications, advantages, and challenges of analyzing proteins and peptides using LC-MS/MS in clinical laboratories.

Morphological variation and expressed sequence tags-simple sequence repeats-based genetic diversity of Aspergillus cristatus in Chinese dark tea.

Introduction: Aspergillus cristatus is a homothallic fungus that is used in the natural fermentation process of Chinese Fuzhuan tea and has been linked to the production of bioactive components. However, not much is known about the variations present in the fungus. To understand the variation of the dominant microorganism, A. cristatus, within dark tea, the present study investigated the genetic and morphological diversity of 70 A. cristatus collected across six provinces of China. Methods: Expressed sequence tags-simple sequence repeats (EST-SSR) loci for A. cristatus were identified and corresponding primers were developed. Subsequently, 15 specimens were selected for PCR amplification. Results: The phylogenetic tree obtained revealed four distinct clusters with a genetic similarity coefficient of 0.983, corresponding to previously identified morphological groups. Five strains (A1, A11, B1, D1, and JH1805) with considerable differences in EST-SSR results were selected for further physiological variation investigation. Microstructural examinations revealed no apparent differentiation among the representative strains. However, colony morphology under a range of culture media varied substantially between strains, as did the extracellular enzymatic activity (cellulase, pectinase, protease, and polyphenol oxidase); the data indicate that there are differences in physiological metabolic capacity among A. cristatus strains. Discussion: Notably, JH1805, B1, and A11 exhibited higher enzymatic activity, indicating their potential application in the production of genetically improved strains. The findings provide valuable insights into species identification, genetic diversity determination, and marker-assisted breeding strategies for A. cristatus.

Thyroid stimulating receptor autoantibodies.

Thyroid-stimulating hormone receptor autoantibodies (TRAbs) play a crucial role as pathogenic antibodies in both the diagnosis and management of Graves' disease (GD). GD, an autoimmune disease resulting from a combination of genetic and environmental factors, is the most common cause of hyperthyroidism. With advancements in technology for TRAb detection and the availability of automated commercial kits, TRAb has become an essential clinical laboratory marker for the diagnosis of GD, as well as extra-thyroidal manifestations like Graves' ophthalmopathy (GO). This article provides a comprehensive review of TRAb, encompassing its clinical assays along with its significance in the clinical setting.

Integrated identification-quantification (ID-Quant) workflow utilizing UPLC-QTOF-MS for the therapeutic drug monitoring of multi-component antibiotics without pure standards: Validation using teicoplanin.

The lack of individual pure standard has hampered the application of therapeutic drug monitoring (TDM) for multi-component antibiotics in clinical laboratories. Here, we aimed to develop an integrated identification-quantification (ID-Quant) workflow based on ultra-high-performance liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry (UHPLC-QTOF-MS) to enable the comprehensive determination of all teicoplanin components without needing pure standards. The workflow comprises three steps. First, non-targeted MSE full scanning was used to detect and identify all potential ingredients. Then, characteristic product ions were selected to generate a quantitative time-of-flight multiple reaction monitoring (Tof-MRM) method. Finally, the constituent composition of teicoplanin injection was determined and utilized as an alternative reference standard to monitor the teicoplanin ingredients in human serum samples. As a result, nine teicoplanin analogs were identified from teicoplanin injection (Sanofi-Aventis, France). The overall performance of the Tof-MRM method was satisfactory in terms of linearity, precision, accuracy, and limits of detection. Utilizing the drug as standard, the individual concentrations for each component in patient serum were determined to be 0.120 µg/mL (A3-1), 0.020 µg/mL (N-1), 0.550 µg/mL (N-2), 0.730 µg/mL (A2-1), 4.26 µg/mL (A2-2,3), 4.79 µg/mL (A2-4,5), and 0.290 µg/mL (N-3), respectively. The distribution pattern of teicoplanin components was also discovered to differ from that in the drug injection. Overall, this integrated ID-Quant workflow based on UHPLC-QTOF-MS enables the robust quantitation of all teicoplanin analogs without the need for individual pure standard. This approach could help address the standard unavailability problem in the TDM of multi-component antibiotics.

Targeted metabolomics profiling in pregnancy associated with vitamin D deficiency.

BACKGROUND: Vitamin D deficiency is common in pregnancy, however, its effects has not been fully elucidated. Here, we conducted targeted metabolomics profiling to study the relationship. METHODS: This study enrolled 111 pregnant women, including sufficient group (n = 9), inadequate group (n = 49) and deficient group (n = 53). Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS)-based targeted metabonomics were used to characterize metabolite profiles associated with vitamin D deficiency in pregnancy. RESULTS: Many metabolites decreased in the inadequate and deficient group, including lipids, amino acids and others. The lipid species included fatty acyls (FA 14:3, FA 26:0; O), glycerolipids (MG 18:2), glycerophospholipids (LPG 20:5, PE-Cer 40:1; O2, PG 29:0), sterol lipids (CE 20:5, ST 28:0; O4, ST 28:1; O4). Decreased amino acids included aromatic amino acids (tryptophan, phenylalanine, tyrosine) and branched-chain amino acids (valine, isoleucine, leucine), proline, methionine, arginine, lysine, alanine, L-kynurenine,5-hydroxy-L-tryptophan, allysine. CONCLUSIONS: This targeted metabolomics profiling indicated that vitamin D supplementation can significantly affect lipids and amino acids metabolism in pregnancy.

Applications of tandem mass spectrometry (MS/MS) in antimicrobial peptides field: Current state and new applications.

Antimicrobial peptides (AMPs) constitute a group of small molecular peptides that exhibit a wide range of antimicrobial activity. These peptides are abundantly present in the innate immune system of various organisms. Given the rise of multidrug-resistant bacteria, microbiological studies have identified AMPs as potential natural antibiotics. In the context of antimicrobial resistance across various human pathogens, AMPs hold considerable promise for clinical applications. However, numerous challenges exist in the detection of AMPs, particularly by immunological and molecular biological methods, especially when studying of newly discovered AMPs in proteomics. This review outlines the current status of AMPs research and the strategies employed in their development, considering resent discoveries and methodologies. Subsequently, we focus on the advanced techniques of mass spectrometry for the quantification of AMPs in diverse samples, and analyzes their application, advantages, and limitations. Additionally, we propose suggestions for the future development of tandem mass spectrometry for the detection of AMPs.

Comparison of 1-stage and 2-stage Managements for Common Bile Duct Stones and Gallstones (CBDS): A Retrospective Study.

OBJECTIVE: The aim of this study was to evaluate the efficacy, safety, and surgical outcomes of 2-stage management, namely preoperative endoscopic retrograde cholangiopancreatography (ERCP) + laparoscopic cholecystectomy (ERCP+LC) or LC + postoperative ERCP (LC+ERCP), as well as 1-stage management, LC + laparoscopic common bile duct exploration (LCBDE) for treating patients with gallstones and common bile duct stones (CBDS). METHODS: This retrospective study analyzed the data of 180 patients with common bile duct stones (CBDS) who were admitted to the Department of General Surgery at Tongji Hospital, Tongji University, between January 2019 and June 2021. The study included 3 groups: ERCP+LC (group 1), LC+ERCP (group 2), and LC+LCBDE (group 3), each consisting of 60 patients. Clinical metrics of the patients were collected and compared among the groups. RESULTS: Group 3 had the shortest operation duration and hospital stay compared with group 1 and group 2. In addition, group 3 had the lowest long-term postoperative complications, particularly the recurrence rate of CBDS. The total cost was also the lowest in group 3. Furthermore, patients in group 3 had the lowest postoperative amylase levels. All patients in the study achieved successful stone clearance. There were no significant differences in the conversion to other procedures rate, postoperative alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, and mortality among the three groups (P > 0.05). CONCLUSIONS: Both 1-stage management and 2-stage management are effective treatments for CBDS. The LC+LCBDE management is a safe treatment option, offering shorter hospital stays and operation duration, lower costs, and fewer complications.

Plasma Steroid Profiling Combined With Machine Learning for the Differential Diagnosis in Mild Autonomous Cortisol Secretion From Nonfunctioning Adenoma in Patients With Adrenal Incidentalomas.

OBJECTIVE: To assess the diagnostic value of combining plasma steroid profiling with machine learning (ML) in differentiating between mild autonomous cortisol secretion (MACS) and nonfunctioning adenoma (NFA) in patients with adrenal incidentalomas. METHODS: The plasma steroid profiles data in the laboratory information system were screened from January 2021 to December 2023. EXtreme Gradient Boosting was applied to establish diagnostic models using plasma 24-steroid panels and/or clinical characteristics of the subjects. The SHapley Additive exPlanation (SHAP) method was used for explaining the model. RESULTS: Seventy-six patients with MACS and 86 patients with NFA were included in the development and internal validation cohort while the external validation cohort consisted of 27 MACS and 21 NFA cases. Among 5 ML models evaluated, eXtreme Gradient Boosting demonstrated superior performance with an area under the curve of 0.77 using 24 steroid hormones. The SHAP method identified 5 steroids that exhibited optimal performance in distinguishing MACS from NFA, namely dehydroepiandrosterone, 11-deoxycortisol, 11ß-hydroxytestosterone, testosterone, and dehydroepiandrosteronesulfate. Upon incorporating clinical features into the model, the area under the curve increased to 0.88, with a sensitivity of 0.77 and specificity of 0.82. Furthermore, the results obtained through SHAP revealed that lower levels of testosterone, dehydroepiandrosterone, low-density lipoprotein cholesterol, body mass index, and adrenocorticotropic hormone along with higher level of 11-deoxycortisol significantly contributed to the identification of MACS in the model. CONCLUSIONS: We have elucidated the utilization of ML-based steroid profiling to discriminate between MACS and NFA in patients with adrenal incidentalomas. This approach holds promise for distinguishing these 2 entities through a single blood collection.

Asymptomatic elevation of parathyroid hormone levels by antibodies against reagent alkaline phosphatase.

CONTEXT: Although immunoassay interference is a well-known phenomenon, its detection in routine clinical practice remains challenging. Most immunoassay interference can be attributed to the presence of heterophilic or anti-hormone antibodies. However, reports on immunoassay interference specifically related to parathyroid hormone (PTH) are scarce. CASE DESCRIPTION: A 77-year-old woman with hypertension, nephrotic syndrome, and high PTH levels for one year was admitted to our Surgical Department for treatment. The patient had no specific symptoms and normal calcium and alkaline phosphatase (ALP) levels but markedly elevated PTH levels. PTH was 2172 pg/mL using the Beckman Coulter system, whereas the Roche, Abbot, and Siemens systems yielded normal results. PTH concentration decreased to 63.8 pg/mL after pretreatment with polyethylene glycol 6000 and did not decrease to normal levels following pretreatment with heterophilic blocking tube-50 (HBT-50), heterophilic blocking reagent (HBR)-21, or HBR-25. When the HBR-21 concentration was increased, serum PTH decreased to 99.0 pg/mL. After treatment with scavenger bovine alkaline phosphatase (inactive), the concentration of PTH decreased to a normal value (51.3 pg/mL). Additionally, PTH (1-84) concentration was 17.6 pg/mL using LC-MS/MS. CONCLUSION: PTH was falsely evaluated due to anti-bovine ALP antibodies (antibodies against reagent ALP). Anti-bovine ALP antibodies should be considered in assays that use ALP as a signal generator.

Steroid profiling in adrenal disease.

The measurement of steroid hormones in blood and urine, which reflects steroid biosynthesis and metabolism, has been recognized as a valuable tool for identifying and distinguishing steroidogenic disorders. The application of mass spectrometry enables the reliable and simultaneous analysis of large panels of steroids, ushering in a new era for diagnosing adrenal diseases. However, the interpretation of complex hormone results necessitates the expertise and experience of skilled clinicians. In this scenario, machine learning techniques are gaining worldwide attention within healthcare fields. The clinical values of combining mass spectrometry-based steroid profiles analysis with machine learning models, also known as steroid metabolomics, have been investigated for identifying and discriminating adrenal disorders such as adrenocortical carcinomas, adrenocortical adenomas, and congenital adrenal hyperplasia. This promising approach is expected to lead to enhanced clinical decision-making in the field of adrenal diseases. This review will focus on the clinical performances of steroid profiling, which is measured using mass spectrometry and analyzed by machine learning techniques, in the realm of decision-making for adrenal diseases.

Evolution of LC-MS/MS in clinical laboratories.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has attracted significant attention in clinical practice owing to its numerous advantages. However, the widespread adoption of this technique is hindered by certain limitations, such as inappropriate analyte selection, low levels of automation, and a lack of specific reference intervals and quality control programs. This review comprehensively summarizes the current challenges associated with LC-MS/MS and proposes potential resolutions. The principle of utility should guide the selection of biomarkers, prioritizing their practical value over sheer quantity. To achieve full-process automation, methodological innovation is crucial for developing high-throughput equipment. Establishing reference intervals for mass spectrometry-based assays across multiple centers and diverse populations is essential for accurate result interpretation. Additionally, the development of commercial quality control materials assumes pivotal importance in ensuring assay reliability and reproducibility. Harmonization and standardization efforts should focus on the development of reference methods and materials for the clinical use of LC-MS/MS. In the future, commercial assay kits and laboratory-developed tests (LDTs) are expected to coexist in clinical laboratories, each offering distinct advantages. The collaborative efforts of diverse professionals is vital for addressing the challenges associated with the clinical application of LC-MS/MS. The anticipated advancements include simplification, increased automation, intelligence, and the standardization of LC-MS/MS, ultimately facilitating its seamless integration into clinical routines for both technicians and clinicians.

A comprehensive LC-MS/MS method for the simultaneous measurement of 24 adrenal steroids: From research to clinical practice.

Steroids are essential in the differential diagnosis of congenital adrenal hyperplasia (CAH) subtypes; however, they may confuse physicians with multifarious results. In this study, we established a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous measurement of 24 steroids and developed a steroid metabolite pathway-based report to aid physicians in understanding these results. Solid-phase extraction was used to concentrate and purify target plasma steroids. The linearity, precision, recovery, and matrix effects were thoroughly evaluated. PowerBuilder was used to transfer the results from LC-MS/MS to the graphic report in a laboratory information management system (LIS) and was applied to different subtypes of CAH. Twenty-four steroids were separated and analyzed in one sample preparation and two injections using LC-MS/MS. The linearity of the steroids was excellent, with coefficients of linear regression greater than 0.99. The relative recovery ranged from 90.0 to 107.1 %, whereas the intra- and total coefficient variations were 1.6 âˆ¼ 8.7 % and 2.0 âˆ¼ 9.9 %, respectively. Matrix effects were compensated after internal standard correction. A graphic combination report mode was established and used to effectively identify CAH subtypes. In conclusion, a useful LC-MS/MS method and graphic combination report of 24 steroids based on their metabolite pathways were established.