Journal of Applied Laboratory Medicine最新文献

Background: The clinical management of chronic myeloid leukemia (CML) patients requires the identification of the type of BCR::ABL1 transcript at diagnosis and the monitoring of its expression and potential tyrosine kinase inhibitor (TKI) resistance mutations during treatment. Detection of resistant mutation requires transcript type-specific amplification of BCR::ABL1 from RNA.

Methods: In this study, a custom RNA-based next-generation sequencing (NGS) assay (Dup-Seq BCR::ABL1) that enables (a) the identification of BCR::ABL1 transcript type and (b) the detection of resistance mutations from common and atypical BCR::ABL1 transcript types was developed and validated. The assay design covers BCR exon 1 to ABL1 exon 10 and employs duplicate PCR amplification for error correction. The custom data analysis pipeline enables breakpoint determination and overlapped mutation calling from duplicates, which minimizes the low-level mutation artifacts.

Results: This study demonstrates that this novel assay achieves high accuracy (positive percent agreement (PPA) for fusion: 98.5%; PPA and negative percent agreement (NPA) for mutation at 97.8% and 100.0%, respectively) and sensitivity (limit of detection (LOD) for mutation detection at 3% from 10 000 copies of BCR::ABL1 input).

Conclusions: The Dup-Seq BCR::ABL1 assay not only allows for the identification of BCR::ABL1 typical and atypical transcript types and accurate and sensitive detection of TKI-resistant mutations but also simplifies molecular testing work flow for the clinical management of CML patients.

Background: A dual filtration-based method for determination of serum labile bound copper (LBC) and LBC fraction (LBC/total copper) was developed. Reduced total copper, elevated LBC, and elevated LBC fraction have been reported in Wilson disease (WD).

Methods: To evaluate the diagnostic performance of these markers, samples were obtained from 21 WD treatment-naïve (WD-TN, no WD treatment or <28 days of treatment) patients, 46 WD standard-of-care-treated (WD-SOC) patients, along with 246 patients representing other potential disorders of copper status. These were then compared to 213 reference interval population patients.

Results: Receiver operating characteristic curves for the reference population vs WD-TN yielded areas under the curve for total copper, LBC, and LBC fraction, of 0.99, 0.81, and 0.98, respectively. Using Youden cutoffs, sensitivity/specificity for WD-TN was 95%/97% for total copper, 71%/85% for LBC, and 95%/94% for LBC fraction. LBC values, but not total copper and LBC fraction, differed substantially between WD-TN and WD-SOC cohorts.We propose a dual model wherein total copper and LBC fraction results must agree to be classified as a "positive" or "negative" result for WD. This correctly classified 19/21 WD-TN patients as positive, and 194/213 reference interval patients as negative. The remaining "indeterminate" patients (representing approximately 9% of the reference and the WD-TN populations) exhibited conflicting total copper and LBC fraction results. When indeterminate results are excluded, this model exhibited apparent 100% sensitivity/specificity.

Conclusions: Agreement of total serum copper and LBC fraction classification may constitute an effective "rule-in" and "rule-out" assessment for WD-TN patients.

Background: We evaluated the Vitros® Immunodiagnostic Products N-terminal pro B-type natriuretic peptide (NT-proBNP) II assay for aiding in diagnosis of heart failure (HF) in patients with acute dyspnea.

Methods: Serum concentrations of NT-proBNP were measured in patient samples from 20 emergency departments across the United States. Study endpoints included sensitivity, specificity, likelihood ratios, and predictive values for diagnosis of acute HF according to age-stratified cutoffs (450, 900, and 1800 pg/mL), and a rule-out age-independent cutoff (300 pg/mL). Additional measures were area under the curve (AUC) for receiver operating characteristic (ROC) curves. Results were also interpreted in patient subgroups with relevant comorbidities, and gray zone/intermediate assay values.

Results: Of 2200 patients, 1095 (49.8%) were diagnosed with HF by clinical adjudication. Sensitivity and specificity for Vitros NT-proBNP II ranged from 84.0% to 92.1%, and 81.4% to 86.5%, respectively, within and across age groups, and positive predictive values were 80.4% to 85.7%. Using the rule-out cutoff, the negative predictive value was 97.9%, with a negative likelihood ratio of 0.02. In subgroups with comorbidities potentially affecting NT-proBNP concentrations, sensitivities ranged from 82.6% to 89.5%, and AUCs for ROC curves were 0.899 to 0.915.

Conclusions: The Vitros NT-proBNP II assay demonstrated excellent clinical performance using age-stratified cutoffs along with other clinical information for supporting diagnosis of HF, and can rule out HF with a high negative predictive value using the age-independent cutoff. The assay retained utility in patient subgroups with conditions that influence NT-proBNP concentration, and for those with gray zone results.

Clinicaltrials.gov registration number: NCT03548909.

Background: Current methods for evaluating liver health rely on nonspecific blood tests, elastography surrogates for fibrosis, and invasive procedures, none of which directly measure liver function and physiology. Herein we present the analytical validation of a unique, highly sensitive LC-MS/MS assay and dual-sample oral (DuO) cholate challenge test to reliably quantify serial serum concentrations of cholate isotopes administered to patients with liver diseases. The clearance of administered cholate isotopes measured by the assay provides information about liver function and physiology.

Methods: Analytical method validation of the cholate assay analytes (endogenous unlabeled cholic acid, 24-13C-cholic acid, and 2,2,4,4-D4-cholic acid) in terms of accuracy, precision, analytical sensitivity, analytical specificity, and range of reliable response was completed in human serum samples spiked with quality controls and calibrators in accordance with applicable guidelines. DuO test parameters were validated using samples from 48 subjects representing various liver disease etiologies.

Results: Accuracy (mean biases) for all analytes ranged from 0.1% to 3.7%. Using a nested components-of-variance design (20 days, 2 runs per day, 2 replicates per sample), total imprecision for all analytes ranged from 2.3% to 8.4%. Lower and upper limits of quantitation were established and validated at 0.1 to 10.0 µM. Matrix effects and potential interferents did not affect assay performance. DuO test validation met all prespecified acceptance criteria.

Conclusions: The method validation studies described herein established the performance characteristics in terms of accuracy, precision, analytical sensitivity, analytical specificity, reportable ranges, and reference intervals of the LC-MS/MS cholate assay and DuO test.

Background: Recently, a major manufacturer recalled several lots of iron assay reagent due to positive bias of roughly 15%-30% and the cause remains unknown. This study investigated the root cause of this positive bias and evaluated a simple practical approach to improve the assay.

Methods: Performance comparison of recalled and unimpacted iron assay kits was done utilizing calibrators, quality control (QC) materials, and 42 remnant patient samples. Spectral scan and trace elements analysis of R1 and R2 reagents was performed. Copper (Cu) and thiourea (TU) spiking experiments were utilized to elucidate the cause and prevention of positive bias seen with recalled lots.

Results: Iron measurements in QC materials and patient samples using recalled reagents generated a positive bias of 17.5% and 21%, respectively. Correspondingly, the recalled R2 reagents, but not R1, showed a rise in basal absorbance along with an unanticipated presence of Cu (22.7 µg/dL) and lead (7.5 µg/L). Cu spiking to recalled and unimpacted R2 reagent intensified the reagent color besides falsely increasing its absorbance, calibration factor, and patient iron measurements. Interestingly, addition of TU (65 mmol/L) to R2 reagent from unimpacted lot prevented the short-term and prolonged Cu-induced spurious rise in calibration factor and patient iron estimations.

Conclusions: We conclude that accidental copper contamination of R2 reagent during manufacturing could be a reason underlying the positive bias in the recalled iron reagent lots. Addition of TU in ferene-containing R2 reagent is a simple and effective means to prevent Cu-induced false elevation in iron values.

Background: The presence of anti-interferon (IFN)-α2 autoantibodies is a strong indicator of severe disease course during viral infections and is observed in autoimmune diseases (e.g., myasthenia gravis). Detection of these autoantibodies during severe bacterial infections is understudied. Multiple anti-IFN-α2 autoantibody screening assays are available. However, the results do not always correlate with the neutralizing capacity of the autoantibodies.

Methods: Anti-IFN-α2 antibodies were measured by a Luminex-based assay in serum samples from individuals admitted to the intensive care unit infected with influenza (n = 38), invasive bacteria (n = 152), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (n = 52). Anti-IFN-α2 antibodies were also studied in individuals with myasthenia gravis (n = 22) and in healthy individuals (n = 37). Individuals testing positive by Luminex were subsequently tested by enzyme-linked immunosorbent assay (ELISA) and tested for nonspecific reactivity and neutralization.

Results: Three of 16 Luminex-positive samples had nonspecific reactivity, 11/16 were positive by ELISA, and 10/16 had neutralizing activity. Anti-IFN-α2 antibodies were found in individuals infected with SARS-CoV-2 (7/52), influenza (3/38), invasive bacteria [2/152, of which 1 was Legionella pneumophilia and was 1 Escherichia coli (E. coli) (out of 39 E. coli infections)], and in individuals with myasthenia gravis (2/22).

Conclusions: Anti-IFN-α2 autoantibodies were detected in viral infections, myasthenia gravis, and rarely in bacterial infections. ELISA and Luminex screening assays do not give similar results. Nonspecific reactivity and functional assays are necessary to validate the screening test result.