BACKGROUND:Methylation-specific PCR (MSP) is widely used in cancer diagnostics and requires positive controls to confirm DNA integrity and bisulfite conversion. Positive control assays include conversion-specific PCR (CSP) and cytosine-free fragment (CFF) assays. Despite their ubiquity and critical need in MSP assays, positive control assays lack systematic technical performance evaluation. METHODS:Twelve positive control assays (9 CSP, 3 CFF), including 10 established and 2 novel designs, were evaluated under PCR conditions optimized for modern multiplexed MSP. Assays were tested via DNA-intercalating dye detection across bisulfite-converted and unconverted human DNA templates for amplification consistency across replicates, conversion specificity, primer dimer formation, and PCR efficiency. The 2 novel assays were multiplexed and assessed on cell-free DNA and tissue samples via droplet digital PCR. RESULTS:Performance varied markedly across assays. Four CSP assays (ACTB-C, ACTB-D, ACTB-P3, and ZGPAT) demonstrated low replicate variability (SD 0.1-0.6 Cq), near-ideal PCR efficiencies (98%-102%) with strong linearity (R2 ≥ 0.996), minimal amplification of unconverted blood DNA (≤25%), and limited primer dimer formation. Among CFF assays, cfUQ11-CFF showed the best performance, with efficiencies of 92%-97%, strong linearity (R2 ≥ 0.995), and low rates of primer dimer formation (25% of no-template control wells). In multiplex digital PCR on cfDNA, the 2 novel assays demonstrated strong concordance with Qubit-derived DNA estimates (R2 = 0.928 and 0.912). CONCLUSIONS:Positive control assay selection critically impacts MSP reliability and quantitation. From 12 assays tested, we highlight 4 CSP assays and one CFF assay that meet performance requirements and are suitable for further application.
BACKGROUND:Plasma proinsulin concentrations are used to investigate hypoglycemia. They have also been proposed as a marker of β-cell function, particularly as a ratio with C-peptide. Immunoassays remain the primary method for measuring proinsulin despite potential limitations. Mass spectrometry-based assays have been described, but are semiquantitative or rely on nano-flow liquid chromatography. We aimed to develop a liquid chromatography-tandem mass spectrometry assay (LC-MS/MS) at typical clinical laboratory flow rates to quantify proinsulin and its partially processed forms in human plasma and ensure accuracy over time with distributable well-characterized calibrators. METHODS:Sample preparation consists of protein precipitation, Glu-C digestion, peptide immunoaffinity enrichment, and LC-MS/MS analysis of 2 surrogate peptides: RGFFYTPKTRREAE spans the cleavage site for des-31,32-proinsulin and GSLQKRGIVE spans the site for des-64,65-proinsulin. A purified protein calibration material was characterized (HPLC and amino acid analysis) and used to value-assign a matrix-matched single-point calibration material. RESULTS:Within-batch and between-batch imprecision were ≤12.2% and ≤16.4%, respectively. The assay was linear from 0.14 to 53.5 pM and 1.1 to 91.6 pM, with a lower limit of the measuring interval of 2.8 and 8.8 pM for RGFFYTPKTRREAE and GSLQKRGIVE, respectively. The concentration of des-31,32-proinsulin increased more during childhood than intact proinsulin. Participants with type 1 diabetes-associated autoantibodies had higher proinsulin-to-C-peptide ratios. Method comparison with 2 commercial immunoassays revealed variable cross-reactivity with insulin. CONCLUSION:The validated assay is robust and will be a useful tool for advancing studies of β-cell function. A detailed standard operating procedure, well-characterized calibration material, and monoclonal antibodies are available for adoption in other laboratories.
BACKGROUND:Multiplexed assays, which simultaneously measure many analytes from a single sample, have become increasingly significant for laboratory diagnosis. Many multiplexed assays with application to clinical diagnosis consist of analyte measurements that can fail individually. In these cases, traditional statistical quantitative quality control (QC) measures cannot be used without creating an unacceptably high false rejection rate. METHODS:We developed stochastic simulation software (qcsim) to calculate and visualize the detection power of complex QC rule combinations, including traditional Westgard rules as well as statistical tests of multiple QC repeats, with arbitrary degrees of multiplexing and levels of control. We used this approach to evaluate novel QC models that maintain stringent control of the bias and imprecision of each analyte in a highly multiplexed assay ("panel"). For classifier-based assays that use an algorithm to generate a small number of diagnostic outcomes from a large number of analytes (a "pattern"), we use perturbation analysis to assess the effect of different classifiers with a single analytical platform. RESULTS:Multiple QC approaches are able to overcome the challenge of highly multiplexed assays, and we demonstrate successful strategies that control the false rejection rate using either high analytical performance (low imprecision) or multiple QC replicates. We also demonstrate that, for pattern-based assays, algorithmic details of the specific classifier determine both critical analytes and the required stringency of the QC design. CONCLUSIONS:These results demonstrate multiple QC strategies that control highly multiplexed assays (1000-plex) at a level that is comparable to traditional QC schemes for single-analyte assays.
BACKGROUND:Adrenal-related biomarkers are central in evaluating endocrine causes of secondary hypertension, but their interpretation is influenced by biological variation (BV). In this study, we aimed to estimate the within-subject (CVI) and between-subject (CVG) BV and derive the reference change values (RCVs), the Index of Individuality (II), and the Harris-Brown heterogeneity ratio (HBR) for aldosterone, renin, and the aldosterone-renin ratio (ARR), ACTH, the cortisol/ACTH ratio, metanephrines, and normetanephrines. METHODS:Weekly blood samples were collected from 30 healthy volunteers for 10 consecutive weeks under standardized conditions and were analyzed using state-of-the-art analytical methods. A Bayesian hierarchical model was applied to estimate the posterior distributions of the BV components, yielding estimates of the CVI, CVG, RCVs, II, and HBR. RESULTS:High BV was observed for aldosterone, renin, and the ARR. Aldosterone showed the highest CVI (48.8%), while metanephrine showed the lowest (13.5%). The CVG was high for most measurands, whereas metanephrines showed comparatively low CVG (30.5%). No major sex differences were observed for the biomarkers after performing sex-stratified estimates. The HBR indicated population homogeneity for ARR (female), ACTH (male), and both metanephrines, while aldosterone, renin, ARR (male and mixed), and ACTH (mixed and female) showed heterogeneity. All biomarkers had an II < 1, and RCVs ranged from -72.3% to +261.2% (aldosterone) and -32.9% to +48.9% (metanephrine). CONCLUSIONS:This study provides BV estimates for multiple biomarkers relevant to endocrine secondary hypertension. The results underscore the importance of applying BV-derived tools, including RCVs and II, to improve the interpretation of serial measurements in diagnosis and follow-up.
INTRODUCTION:Noninvasive cell-free DNA (cfDNA) metagenomic sequencing enables hypothesis-free detection of microbial pathogens in patients with suspected infections. However, its clinical sensitivity is often limited by the overwhelming background of host-derived cfDNA, which can obscure low-abundance microbial signals. We developed an epigenetically guided enrichment strategy, termed Epigenetically filtered Metagenomic Sequencing (EpiMeta-seq), to selectively enrich microbial cfDNA based on fundamental differences in DNA methylation between microbial and human genomes. METHODS:EpiMeta-seq uses the methylation-sensitive restriction enzyme HpaII to selectively digest unmethylated CCGG sites, which are prevalent in microbial genomes but largely methylated in human DNA. Only fragments cleaved once at unmethylated sites are incorporated into sequencing libraries, thereby enriching microbial cfDNA prior to sequencing. We assessed plasma samples from patients with microbiologically confirmed infections. Metagenomics informatics involved alignment, removal of host DNA, and taxonomic classification of sequencing reads to a curated reference database. RESULTS:In spike-in experiments at a 1:1000 dilution, EpiMeta-seq achieved a mean enrichment of 24.5-fold for fungal species and 11.4-fold for bacterial species compared with unenriched whole-genome sequencing. In 23 clinical plasma samples representing 12 pathogens, EpiMeta-seq produced an average 10.0-fold increase in microbial reads per million. Viral DNA showed the highest enrichment (mean 11.5-fold), while bacterial enrichment varied across species (1.2- to 30.8-fold). CONCLUSIONS:By leveraging genome-wide methylation differences between host and microbial DNA, EpiMeta-seq is a proof-of-concept, orthogonal enrichment strategy for improving microbial cfDNA signal-to-background ratio across diverse pathogen types in metagenomic sequencing.
BACKGROUND:Therapeutic monoclonal antibodies (t-mAbs) are widely used in oncology, hematology, and immune-mediated diseases. Some t-mAbs are detectable by serum protein electrophoresis (SPEP) and immunofixation (IF), potentially mimicking monoclonal gammopathies. Despite widespread use, migration profiles of many t-mAbs remain incompletely described. METHODS:Thirty-six t-mAbs were tested using Sebia Capillarys 3 (SPEP) and Hydrasis (IF) systems. Normal human sera, free of electrophoretic abnormalities, were spiked with each t-mAb at its reported maximum therapeutic concentration (Cmax). Electropherogram data were processed with a Python-based workflow for axis alignment, standardized profile overlay, and visualization. T-mAbs not detected at Cmax were assessed at 500 mg/L to determine their electrophoretic migration profiles. RESULTS:No interference was observed below 110 mg/L. All t-mAbs tested above this threshold were detectable by SPEP and IF. Migration patterns were newly characterized for several t-mAbs, including bispecific and drug-conjugated antibodies, and confirmed for previously described agents. Overlay analysis enabled refined attribution of migration zones within alpha and beta regions. T-mAbs detectability was summarized in a consolidated table. A stepwise decision algorithm was developed to guide interpretation of suspected t-mAb-related interference. CONCLUSION:This study expands knowledge of t-mAb interference with SPEP and IF and provides standardized electrophoretic profiles for established and emerging therapies. The summary table and decision algorithm offer practical tools for clinical laboratories, facilitating interpretation of electrophoretic patterns, supporting differentiation between t-mAb and monoclonal gammopathies, and highlighting the need for continued characterization of novel t-mAbs entering routine practice.
BACKGROUND:The quantification of plasma glucagon and oxyntomodulin is important in the assessment of α-cell function, which is impaired in patients with diabetes. We aimed to transfer between laboratories a novel assay that uses liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the sensitive and specific measurement of these highly homologous hormones. METHODS:Simultaneous measurement of glucagon and oxyntomodulin used immunoaffinity enrichment and LC-MS/MS. Immunoenrichment used monoclonal antibodies that are available at-cost to researchers (deposited at the Developmental Studies Hybridoma Bank). Pure synthetic glucagon, characterized for purity and concentration, was used as a calibrant and is available to others. A detailed standard operating procedure was shared between 3 laboratories and the performance of the method was evaluated with samples collected from patients with and without diabetes. Method comparison was made with 2 commercially available, FDA-registered glucagon immunoassays. RESULTS:The method was linear over the normal range (1-20 pM). When measured in duplicate, the median interlaboratory imprecision (%CV) of the measurement of 40 samples was 6.3% (IQR 4.5%) and 14.4% (IQR 12.4%) for glucagon and oxyntomodulin, respectively. Method comparison with commercially available immunoassays demonstrated good (Mercodia, R = 0.92) or fair (Ansh, R = 0.70) agreement. Multivariable linear regression using LC-MS/MS glucagon and oxyntomodulin concentrations to predict immunoassay results indicated significant cross-reactivity of each immunoassay with oxyntomodulin. CONCLUSION:We have validated a sensitive and specific assay for glucagon and oxyntomodulin that can be deployed in high-complexity clinical laboratories for research or the care of patients. Commercially available glucagon immunoassays have significant interference from molecules other than glucagon.
BACKGROUND:Comprehensive drug testing (CDT) by liquid chromatography-high-resolution mass spectrometry (LC-HRMS) is a valuable method for evaluating clinical samples for unknown toxicological agents. However, the sensitivity of CDT methods is generally lower than targeted approaches. A novel MS technology, linear ion trap (LIT)-pulsing, has demonstrated improved sensitivity in proteomics and metabolomics applications, but its utility and interactions with different HRMS acquisition types, such as information-dependent acquisition (IDA) and sequential window acquisition of all theoretical fragment ion spectra (SWATH), have not been explored in small molecule analysis. METHODS:CDT methods utilizing both IDA and SWATH acquisition were developed on a quadrupole time-of-flight (QTOF) instrument featuring LIT pulsing. Limits of detection (LODs) and process efficiencies were estimated for 150 toxicological agents. Additionally, 101 patient urine specimens were used to compare both methods against an established clinical CDT without LIT-pulsing. Selectivity was assessed using a targeted LC-HRMS method. RESULTS:LIT-pulsed SWATH acquisition improved detection of toxicological agents compared to LIT-pulsed IDA, with a median 5-fold reduction to measured LODs and an average 5.9% reduction to matrix suppression. In authentic patient samples, LIT-pulsed IDA and SWATH acquisition identified 889 and 1083 toxicological agents compared to 835 using IDA without LIT-pulsing. The estimated selectivity of LIT-pulsed IDA was improved compared to conventional IDA acquisition, but SWATH acquisition demonstrated significant nonselectivity. CONCLUSION:LIT-pulsing improves the sensitivity and selectivity of CDT methods. The largest improvements to sensitivity are observed using SWATH acquisition; however, these gains are accompanied by reduced selectivity. This highlights the need for careful validation of SWATH CDT methods to ensure high clinical performance.
