Head-to-head comparison of four plasma neurofilament light chain (NfL) immunoassays.

BACKGROUND: Neurofilament Light Chain (NfL) is an emerging blood biomarker of neuro-axonal injury and neurodegeneration with the potential to be used in the clinical management of various neurological conditions. Various NfL immunoassays are in development on high-throughput automated systems, but little information is available related to the comparability between assays. In this study, we performed a head-to-head comparison of four NfL immunoassays using plasma samples from individuals with various neurological conditions. METHODS: EDTA plasma samples in which NfL was ordered clinically were stratified according to diagnosis. NfL concentrations (pg/mL) in plasma were obtained using the Quanterix Simoa®, the Roche Elecsys, the Siemens Healthineers Atellica®IM, and the Fujirebio Lumipulse® NfL assays. Passing-Bablok regression analyses were performed to assess the correlation and bias between methods. Additionally, the distribution of NfL concentrations for each assay was assessed in three disease groups: amyotrophic lateral sclerosis (ALS) upon initial diagnosis, ALS treated, and multiple sclerosis (MS). RESULTS: The R2 between assays were all ≥ 0.95, however, significant proportional bias was observed between some assays. In particular, the Roche Elecsys assay NfL concentrations were significantly lower (∼85 %) when compared against the other three assays. The four assays were comparable with regards to the percentage of patients that were identified as having an elevated NfL result in the various clinical groups: ALS initial diagnoses (83-94 %), ALS untreated (93-100 %), and MS (8-18 %). CONCLUSIONS: This is the first study describing a head-to-head comparison of four automated NfL immunoassays. We demonstrate that there is a strong correlation between assays but a lack of standardization which is evident by the bias observed between some of the evaluated methods. These analytical differences will be important to consider when using NfL as a biomarker of neurodegeneration.

Biological variation estimates for serum neurofilament light chain in healthy subjects.

OBJECTIVES: Neurofilament light chain (NfL) is an emerging biomarker of neurodegeneration disorders. Knowledge of the biological variation (BV) can facilitate proper interpretation between serial measurements. Here BV estimates for serum NfL (sNfL) are provided. METHODS: Serum samples were collected weekly from 24 apparently healthy subjects for 10 consecutive weeks and analyzed in duplicate using the Siemens Healthineers sNfL assay on the Atellica® IM Analyzer. Outlier detection, variance homogeneity analyses, and trend analysis were performed followed by CV-ANOVA to determine BV and analytical variation (CVA) estimates with 95%CI and the associated reference change values (RCV) and analytical performance specifications (APS). RESULTS: Despite observed differences in sNfL concentrations between males and females, BV estimates remained consistent across genders. Both within-subject BV (CVI) for males (10.7%, 95%CI; 9.2-12.6) and females (9.1%, 95%CI; 7.8-10.9) and between-subject BV (CVG) for males (26.1%, 95%CI; 18.0-45.6) and females (30.2%, 95%CI; 20.9-53.5) were comparable. An index of individuality value of 0.33 highlights significant individuality, indicating the potential efficacy of personalized reference intervals in patient monitoring. CONCLUSIONS: The established BV estimates for sNfL underscore its potential as a valuable biomarker for monitoring neurodegenerative diseases, offering a foundation for improved decision-making in clinical settings.

Functional Layer-by-Layer Thin Films of Inducible Nitric Oxide (NO) Synthase Oxygenase and Polyethylenimine: Modulation of Enzyme Loading and NO-Release Activity.

Nitric oxide (NO) release counteracts platelet aggregation and prevents the thrombosis cascade in the inner walls of blood vessels. NO-release coatings also prevent thrombus formation on the surface of blood-contacting medical devices. Our previous work has shown that inducible nitric oxide synthase (iNOS) films release NO fluxes upon enzymatic conversion of the substrate l-arginine. In this work, we report on the modulation of enzyme loading in layer-by-layer (LbL) thin films of inducible nitric oxide synthase oxygenase (iNOSoxy) on polyethylenimine (PEI). The layer of iNOSoxy is electrostatically adsorbed onto the PEI layer. The pH of the iNOSoxy solution affects the amount of enzyme adsorbed. The overall negative surface charge of iNOSoxy in solution depends on the pH and hence determines the density of adsorbed protein on the positively charged PEI layer. We used buffered iNOSoxy solutions adjusted to pHs 8.6 and 7.0, while saline PEI solution was used at pH 7.0. Atomic force microscopy imaging of the outermost layer shows higher protein adsorption with iNOSoxy at pH 8.6 than with a solution of iNOSoxy at pH 7.0. Graphite electrodes with PEI/iNOSoxy films show higher catalytic currents for nitric oxide reduction mediated by iNOSoxy. The higher enzyme loading translates into higher NO flux when the enzyme-modified surface is exposed to a solution containing the substrate and a source of electrons. Spectrophotometric assays showed higher NO fluxes with iNOSoxy/PEI films built at pH 8.6 than with films built at pH 7.0. Fourier transform infrared analysis of iNOSoxy adsorbed on PEI at pH 8.6 and 7.0 shows structural differences of iNOSoxy in films, which explains the observed changes in enzymatic activity. Our findings show that pH provides a strategy to optimize the NOS loading and enzyme activity in NOS-based LbL thin films, which enables improved NO release with minimum layers of PEI/NOS.

Development of a fast and simple liquid chromatography-tandem mass spectrometry method for the quantitation of argatroban in patient plasma samples.

An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the direct measurement of argatroban in human plasma was developed and compared with the activity-based Hemoclot Thrombin Inhibitors assay. UPLC-MS/MS was performed using diclofenac as an internal standard. In summary, argatroban and diclofenac were extracted from 100 µL of plasma using a methanol precipitation protocol, and chromatographic separation was performed on an ACQUITY TQD mass spectrometer using a UPLC C18 BEH 1.7 µm column with a water and methanol gradient containing 0.1% formic acid. The detection and quantitation were performed using positive ion electrospray ionization and multiple reaction monitoring (MRM) mode. The UPLC-MS/MS method was linear over the concentration range of 0.003-3.0 µg/mL, with a lower limit of quantitation for argatroban of 0.003 µg/mL. The intra- and inter-assay imprecision was less than 12% at the plasma argatroban concentrations tested. Good correlation was demonstrated between the UPLC-MS/MS method and the indirect activity-based assay for determination of argatroban. However, increased plasma fibrinogen levels caused underestimation of argatroban levels using the indirect activity-based assay, whereas the UPLC-MS/MS method was unaffected. UPLC-MS/MS provides a relatively simple, sensitive, and rapid means of argatroban monitoring. It has successfully been applied to assess plasma argatroban concentrations in hospitalized patients and may provide a more accurate determination of argatroban concentrations than an activity-based assay in certain clinical conditions.

Plasma and cerebral spinal fluid tranexamic acid quantitation in cardiopulmonary bypass patients.

A method for the determination of tranexamic acid (TXA) in human plasma and cerebral spinal fluid (CSF) was developed. Analyses were performed by ultra performance liquid chromatography with tandem mass spectrometry detection (UPLC-MS/MS) using ɛ-aminocaproic acid (ACA) as an internal standard. TXA and ACA were extracted from a 50 µL sample of plasma or CSF using a methanol protein crash protocol, and chromatographic separation was performed on an ACQUITY™ TQD mass spectrometer using a UPLC C18 BEH 1.7 µm column with a water and methanol gradient containing 0.1% formic acid. The detection and quantitation was performed by positive ion electrospray ionization using the multiple reaction monitoring (MRM) mode. The method was linear over the concentration range of 0.1-10.0 µg/mL, with lower limit of quantitation of 0.1 µg/mL for TXA. The intra- and inter-assay precision was less than 12% and 13% respectively at the plasma and CSF TXA concentrations tested. The present method provides a relatively simple and sensitive assay with short turn-around-time. The method has been successfully applied to assess the plasma and CSF concentrations of tranexamic acid achieved with only one dosing regimen of tranexamic acid in patients undergoing cardiopulmonary bypass surgery (CPB).