Clinical chemistry and laboratory medicine最新文献

Objectives: Diagnosing atypical IgG4-mediated anti-glomerular basement membrane (anti-GBM) disease is challenging because conventional serological assays poorly detect IgG4 antibodies. Here we study which commercial assays are affected and we describe proof-of-concept of improved IgG4 anti-GBM detection.

Methods: To investigate the scope of the diagnostic dilemma detecting IgG4 anti-GBM antibodies, serum from a patient with atypical IgG4-mediated anti-GBM was distributed to 36 laboratories participating in the Dutch External Quality Assessment (EQA) program. To improve IgG4 anti-GBM detection in the fluorescent enzyme immunoassay (FEIA), the standard anti-IgG conjugate was replaced with anti-IgG4 conjugate.

Results: We report the diagnostic delay of a patient with atypical anti-GBM disease who presented with an indolent disease course. Histopathology comprised hallmarks of classic anti-GBM disease including bright linear IgG deposits along the GBM but without the typical findings of diffuse crescentic and necrotizing glomerulonephritis. IgG anti-GBM test results were repeatedly negative. Histopathological subclass analysis demonstrated that the linear IgG deposits were predominantly IgG4. All 36 Dutch EQA-participants reported negative anti-GBM test results, demonstrating that the diagnostic challenge of detecting IgG4 anti-GBM is broadly applicable across multiple diagnostic assays. A minor modification to the manufacturer's standard protocol of the FEIA anti-GBM dramatically improved the assay's performance for measuring antibodies of the IgG4 isotype. Using the modified IgG4-GBM test, we were able to diagnose and monitor one more patient with atypical IgG4-mediated anti-GBM disease.

Conclusions: Here we demonstrate proof-of-concept of a modified IgG4 anti-GBM blood test allowing serological confirmation of atypical anti-GBM disease and sensitive monitoring of therapy response.

Objectives: Newborn screening for congenital hypothyroidism (CH) relies on thyroid-stimulating hormone (TSH) levels from dried blood spots (DBS). We investigated whether incorporating TSH variation across serial DBS could improve prediction of CH in term and preterm infants with complex risk profiles.

Methods: Among 207,895 newborns screened, 272 (0.13 %) were diagnosed with CH. TSH variations across 3 serial DBS were analyzed in 6,146 healthy infants (2.96 %). Predictive algorithms were developed using linear mixed-effects models in 1,968 term and 1,387 preterm infants with ≥2 and ≥3 DBS, respectively. Average DBS collection times were 58, 260, and 478 h for term and 63, 350, and 664 h for preterm infants. TSH was measured by GSP neonatal hTSH assay (Revvity).

Results: Daily TSH variation was influenced by the initial DBS1 value. For DBS1 >5.2 and <1.7 mUI/L, increase and decrease, respectively, in TSH level on DBS2 is detectable, and this particularly occurs in preterm infants. In preterms, CH could be excluded when TSH remained <5 mUI/L on DBS1, <6 mUI/L on DBS2, <4 mUI/L on DBS3, and with daily variation <12 % from DBS1 to DBS3 (sensitivity 100 %; specificity 77.85 %). Term infants with TSH <11 mUI/L on DBS1, <4.5 mUI/L on DBS2, and daily variation <13 % from DBS1 to DBS2 may be ruled out for CH (sensitivity 96.5 %; specificity 66.6 %).

Conclusions: Distinct predictive algorithms for term and preterm newborns, incorporating TSH variations as daily percentage changes, may improve CH rule out in children with complex risk profiles.

Objectives: Accurate B-lineage assignment in acute leukemia is critical for therapeutic decisions. While nuclear expression of PAX5 protein (nPAX5) is a highly reliable marker for B-cell differentiation, its conventional assessment by time-consuming immunohistochemistry (IHC) is not ideal for rapid diagnosis. This study aimed to validate the utility of flow cytometric (FCM) detection of nPAX5 for the diagnosis of B-lymphoblastic leukemia (B-ALL), potentially enhancing diagnostic efficiency.

Methods: A retrospective study was conducted, comparing nPAX5 expression by FCM and IHC in 125 bone marrow biopsies (57 B-ALL, 12 T-ALL, 56 AML). Further diagnostic performance analysis, using ROC analysis of the geometric mean fluorescence intensity ratio (Blast/Normal T-cell), was performed for nPAX5, cCD22, and cCD79a across a larger cohort of 538 acute leukemia cases (123 B-ALL, 29 T-ALL, 386 AML).

Results: FCM and IHC for PAX5 expression showed 100 % concordance across the 125 cases (57/57 B-ALL positive; 0/12 T-ALL negative). The single PAX5-positive AML case harbored the t(8;21) translocation. ROC analysis demonstrated excellent diagnostic performance for both cCD79a (AUC 0.980) and nPAX5 (AUC 0.955). At optimal criteria, cCD79a achieved 100.00 % specificity and 91.06 % sensitivity. nPAX5 showed strong utility, matching the 91.06 % sensitivity with 89.45 % specificity (criterion >6.30). cCD22 exhibited moderate discriminatory power (AUC 0.807).

Conclusions: Flow cytometric nuclear PAX5 (nPAX5) demonstrated 100 % agreement with IHC and exhibited excellent diagnostic accuracy (AUC 0.955, 91.06 % sensitivity). These compelling results validate nPAX5 as a reliable and powerful FCM marker, supporting its immediate adoption for rapid and efficient B-lineage assignment in acute leukemia diagnostics.

Objectives: Testing for high-sensitivity cardiac troponin (hs-cTn) often occurs following pediatric cardiac surgery, although evidence regarding its utility remains heterogeneous. This study aimed to assess the prognostic value of postoperative hs-cTnT patterns detected within 48 h after cardiac surgery.

Methods: Serum hs-cTnT (Roche Diagnostics, 5th generation assay) concentrations were measured post-operatively at three time points: upon pediatric intensive care unit (PICU) admission (T1), 13 h after admission (T2), and 24 h after T2 (T3). Surgical and postoperative variables were recorded. The outcome was a composite of 30-day mortality and/or PICU stay >10 days. Multivariable logistic regression and model discrimination were evaluated.

Results: Over 15 months, 154 patients (56.5 % male) with a median age of 3.8 (25th-75th percentile: 1.2-7.8) months and a median Risk Assessment for Congenital Heart Surgery score of 2, were included in this study. The outcome occurred in 24 % of the population (with the 30-day mortality rate being 7.8 %). The time point of the highest recorded hs-cTnT concentration, and not the concentration, was significantly associated with the outcome (p=0.001). Patients, whose peak hs-cTnT concentration occurred later after surgery had a sixfold higher risk of the adverse outcome. Urgent/emergent procedures were associated with a 3.6-fold increase in relative risk, and each additional minute of cardiopulmonary bypass (CPB) time conferred a 1.6 % incremental increase in risk. The multivariate model including time to troponin peak, CPB duration, need for urgent/emergent procedures showed a good discriminatory ability (c-statistic=0.84; 95 %CI: 0.78-0.90).

Conclusions: Timing of hs-cTnT elevation post cardiac surgery is a valuable prognostic marker in children.

Objectives: The reliability of cerebrospinal fluid (CSF) and serum biomarkers depends on sample integrity, which is strongly influenced by storage conditions. While -80 °C is considered the gold standard for long-term preservation, it requires specialized infrastructure, whereas -20 °C is more accessible but may compromise protein stability. This study aimed to evaluate the stability of three clinically relevant biomarkers - neuron-specific enolase (NSE), S100, and β-2 microglobulin (B2M), associated with neuronal injury and neuroinflammation - in CSF and serum samples stored at -20 °C and -80 °C over different time periods.

Methods: A total of 63 CSF and 56 serum samples from pediatric patients were analyzed. Samples were stored at -20 °C (24 h and 6 months) and -80 °C (6 and 12 months). NSE and S100 were measured by electrochemiluminescence immunoassays and B2M by turbidimetric immunoassay. Biomarker variability was expressed as relative percentage change from baseline. A maximum permissible error of ±15 % for CSF and ±10 % for serum was selected.

Results: NSE and S100 were highly unstable at -20 °C. In CSF, after 6 months, NSE decreased by ∼74 % and S100 by ∼71 %; in serum, NSE declined by ∼54 % and S100 by ∼13 %. Remarkably, after only 24 h at -20 °C, CSF NSE dropped by 61 %. Storage at -80 °C largely preserved these two biomarkers, with declines below ∼15 % over 12 months. B2M levels remained stable under all conditions. The reduction in CSF NSE at -20 °C was strongly correlated with baseline concentrations.

Conclusions: CSF biomarkers NSE and S100 are highly susceptible to degradation at -20 °C, whereas B2M remains relatively stable. Strict adherence to -80 °C storage protocols is essential to ensure the reliability of biomarker-based diagnostics and research.

Objectives: This study aims to explore the biological variability of calcium homeostasis biomarkers, including calcium, phosphate, magnesium, 25-OH vitamin D, 1,25-OH vitamin D, and parathyroid hormone (PTH), and to develop a model that predicts PTH concentrations based on these parameters.

Methods: Thirty healthy volunteers were sampled on a weekly basis over a period of 10 weeks. Each sample was analyzed in duplicate. Statistical methods included estimating within- and between-subject biological variation (CV_I, CV_G) using ANOVA with outlier removal and a Bayesian model. A linear mixed-effects model (LMM) was used to estimate 95 % prediction intervals for PTH as a function of calcium, phosphate, and 25-OH vitamin D in healthy participants. The effect of applying this prediction model in routine practice was estimated using data from the Laboratory Information System.

Results: Estimated CV_I from ANOVA and the Bayesian model was: PTH, 18.0 % and 17.4 %; calcium, 1.7 % and 1.6 %; phosphate, 9.3 % and 9.3 %; 25-OH vitamin D, 4.7 % and 7.2 %; and 1,25-OH vitamin D, 16.2 % and 17.1 %. The LMM indicated the best 95 % prediction interval for PTH included calcium (PTH (pmol/L)=exp(3.46-2.67·ln(Ca (mmol/L)) ± Z·0.29)) and did not improve with phosphate and/or 25-OH vitamin D. Compared with conventional reference intervals, this model flagged 56 % vs. 41.6 % of routine PTH/calcium results, respectively.

Conclusions: Similar CV_I data were obtained using two different methods. A predictive model can be used to predict concentrations for co-regulated biomarkers, potentially enhancing sensitivity in identifying non-physiological results and facilitating clinicians' awareness of conditions affecting calcium homeostasis.

Objectives: Voriconazole is an antifungal agent measured in serum and plasma for therapeutic drug monitoring. This study aimed to develop a robust isotope-dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) reference measurement procedure (RMP) to ensure accurate and reliable quantification in compliance with metrological standards.

Methods: A commercially available voriconazole material, traceable to the International System of Units (SI), characterized by quantitative nuclear magnetic resonance spectroscopy (qNMR), served as the primary reference material. Chromatographic separation was achieved using a Raptor Biphenyl column, and isotope-labeled voriconazole was utilized as the internal standard. The candidate RMP was validated for selectivity, matrix effects, linearity, precision, accuracy, sample stability, and measurement uncertainty.

Results: The method's selectivity was successfully demonstrated, with no interfering signals observed at the retention time of voriconazole. Assessment of matrix effects revealed no significant differences in slopes across native serum, analyte-free human serum, and Li-hep plasma, confirming the absence of matrix-derived interferences. Intermediate precision was ≤3.7 %, repeatability was ≤2.3 %, and accuracy showed a mean bias ranging from -0.7 to 2.7 % across all matrices and concentration levels. Relative bias for the lower limit of the measuring interval (LLMI) was 1.7 %, with a coefficient of variation (CV) for intermediate precision of 4.3 %. Expanded measurement uncertainties (k=2) for target value assignment (n=6) ranged from 1.4 to 3.4 %.

Conclusions: This validated ID-LC-MS/MS-based RMP proved to be selective, precise, accurate, and reliable for the quantification of voriconazole in human serum and plasma. The RMP demonstrated high accuracy and precision, along with suitable measurement uncertainty, meeting all relevant requirements for an RMP. These findings support the method's suitability for use in establishing target values and ensuring accuracy in clinical laboratories.

Objectives: 5α-dihydrotestosterone (DHT) is a potent androgen, related to male sexual development and irreversibly synthesized from testosterone via 5α-reductase. Dysfunctions in the 5α-reductase system, locally or globally, can have substantial health impacts; measurement of both DHT levels and the testosterone-DHT ratio are thus important for diagnosis and treatment monitoring. For that reason, an isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC MS/MS)-based candidate reference measurement procedure (RMP) to quantify DHT in human serum/plasma was developed.

Methods: We utilized certified primary reference material for DHT provided by the National Measurement Institute of Australia (NMIA) to calibrate our assay and ensure SI (International System of Units) traceability. To mitigate matrix effects and prevent the co-elution of interferences, two-dimensional heart-cut chromatography was employed for LC-MS/MS, in combination with a solid phase extraction (SPE) sample preparation protocol. Selectivity was determined by spiking the prepared internal standard with possibly interfering substances such as the inactive isomer 5β-DHT and other similar compounds. Comparison of standard line slopes was performed to evaluate matrix effects. Precision and accuracy were assessed via a multi-day validation experiment, and variability components estimated using analysis of variance (ANOVA)-based variance component analysis (VCA). Measurement uncertainty (MU) was evaluated in compliance with current guidelines.

Results: This RMP was suitable for analyzing DHT within the range of 0.0160-2.76 ng/mL (0.0551 nmol/L-9.50 nmol/L), demonstrating selectivity, sensitivity and matrix-independence. Intermediate precision was ≤2.1 %, repeatability was ≤1.6 % across all concentration levels, and relative mean bias ranged from -2.2 to 2.5 %, across matrices and concentrations. Expanded MU for reference value assignment (n=6) was ≤2.8 %, irrespective of concentration or sample type.

Conclusions: This RMP exhibited high analytical performance for DHT quantification and met requirements for measurement uncertainty. Additionally, it enabled differentiation between the 5α-DHT and 5β-DHT isomers. Consequently, this RMP is suitable for routine assay standardization and clinical sample evaluation.

Objectives: Dehydroepiandrosterone (DHEA) is a steroid prohormone and important precursor for estrogen and testosterone biosynthesis. For differential diagnosis of sexual development disorders (e.g., polycystic ovary syndrome, congenital adrenal hyperplasia), accurate measurement of DHEA in clinical testing is essential. To address that need for high-quality, reproducible assays, an isotope dilution-liquid chromatography (LC)-tandem mass spectrometry (MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of DHEA was developed and validated.

Methods: A certified primary reference material from the National Measurement Institute of Australia (NMIA), was used for DHEA assay calibration and to ensure traceability to the International System of Units (SI). Two-dimensional heart-cut chromatography was employed for LC-MS/MS in combination with a solid phase extraction sample preparation protocol, to mitigate matrix effects and prevent interference coelution. Selectivity was evaluated by spiking either the analyte or internal standard with potential interferents, such as dehydroepiandrosterone-sulfate, testosterone, and similar steroid compounds. For the evaluation of matrix effects, standard line slopes of various matrices were compared. Precision and accuracy were assessed via a multi-day validation experiment, and variability components were estimated using variance component analysis. Measurement uncertainty was evaluated in compliance with current guidelines.

Results: This candidate RMP proved suitable for the analysis of DHEA in the measurement range of 0.0800-36.0 ng/mL (0.277-125 nmol/L). Predefined requirements for sensitivity and selectivity were fully met, and independence from matrix effects was demonstrated successfully. Across all tested concentration levels, intermediate precision was ≤1.5 % and repeatability was ≤1.0 %, while the relative mean bias ranged from -1.1 to 0.1 %. Regardless of DHEA concentration or sample type, the expanded measurement uncertainty for reference value assignment (n=6) was ≤1.7 %.

Conclusions: This isotope dilution-LC-MS/MS-based candidate RMP for DHEA in human serum and plasma met pre-defined analytical performance requirements such as precision, specificity and measurement uncertainty, and showed superior selectivity towards several potential interferents tested. It is suitable for application in clinical sample evaluation and routine assay standardization.