Impact of age on NIS2+™ and other non-invasive blood tests for the evaluation of liver disease and detection of at-risk MASH.

Background & Aims: Robust performance of non-invasive tests (NITs) across ages is critical to assess liver disease among patients with metabolic dysfunction-associated liver disease (MASLD). We evaluated the impact of age on the performance of NIS2+™ vs. other NITs. Methods: An analysis cohort (N = 1,926) with biopsy-proven MASLD was selected among individuals screened for the phase III RESOLVE-IT clinical trial and divided into ≤45, 46-55, 56-64, and ≥65 years groups. To avoid potential confounding effects, a well-balanced cohort (n = 708; n = 177/age group) was obtained by applying a propensity score-matching algorithm to the analysis cohort. Baseline values of biomarkers and NITs were compared across age groups using one-way ANOVA, and the impact of age and histology were compared through three-way ANOVA. The impact of age on NIT performance for the detection of at-risk metabolic dysfunction-associated steatohepatitis (MASH; MASLD activity score [MAS] ≥4 and fibrosis stage [F] ≥2) was also evaluated. Results: Age did not affect the distributions of NIS2+™ and APRI (aspartate aminotransferase-to-platelet ratio index), but significantly (p <0.0001) impacted those of NFS (NAFLD fibrosis score), FIB-4 (Fibrosis-4 index), and Enhanced Liver Fibrosis (ELF™) score. NIS2+™ was the only NIT on which fibrosis and MAS exerted a moderate to large effect. While the impact of fibrosis on APRI was moderate, that of MAS was low. The impact of age on FIB-4 and NFS was larger than that of fibrosis. NIS2+™ exhibited the highest AUROC values for detecting at-risk MASH across age groups, with stable performances irrespective of cut-offs. Conclusions: NIS2+™ was not significantly impacted by age and was sensitive to both fibrosis and MAS grade, demonstrating a robust performance to rule in/out at-risk MASH with fixed cut-offs. Impact and Implications: While metabolic dysfunction-associated steatotic liver disease (MASLD) can affect individuals of all ages, patient age could represent an important confounding factor when interpreting non-invasive test (NIT) results, highlighting the need for reliable and efficient NITs that are not impacted by age and that could be interpreted with fixed cut-offs, irrespective of patient age. We report the impact of age on different well-established NITs - among those tested, only two panels, NIS2+™ and APRI, were not impacted by age and can be used and interpreted independently of patient age. NIS2+™ was also sensitive to both fibrosis and MAS, further confirming its efficiency for the detection of the composite endpoint of at-risk MASH and its potential as a valuable candidate for large-scale implementation in clinical practice and clinical trials.

Validation of an optimised protocol for quantification of microplastics in heterogenous samples: A case study using green turtle chyme.

Quantifying the extent of microplastic (<5 mm) contamination in the marine environment is an emerging field of study. Reliable extraction of microplastics from the gastro-intestinal content of marine organisms is crucial to evaluate microplastic contamination in marine fauna. Extraction protocols and variations thereof have been reported, however, these have mostly focussed on relatively homogenous samples (i.e. water, sediment, etc.). Here, we present a microplastic extraction protocol for examining green turtle (Chelonia mydas) chyme (i.e. ingested material and digestive tract fluid), which is a heterogeneous composite of various organic dietary items (e.g. seagrass, jellyfish) and incidentally-ingested inorganic materials (sediment). Established extraction methods were modified and combined. This protocol consists of acid digestion of organic matter, emulsification of residual fat, density separation from sediment, and chemical identification by Fourier transform-infrared spectroscopy. This protocol enables the extraction of the most common microplastic contaminants>100 µm: polyethylene, high-density polyethylene, (aminoethyl) polystyrene, polypropylene, and polyvinyl chloride, with 100% efficiency. This validated protocol will enable researchers worldwide to quantify microplastic contamination in turtles in a reliable and comparable way. •Optimization of microplastic extraction from multifarious tissues by applying established methods in a sequential manner.•Effective for heterogenous samples comprising organic and inorganic material.

Ingestion of microplastic debris by green sea turtles (Chelonia mydas) in the Great Barrier Reef: Validation of a sequential extraction protocol.

Ocean contamination by plastics is a global issue. Although ingestion of plastic debris by sea turtles has been widely documented, contamination by microplastics (<5mm) is poorly known and likely to be under-reported. We developed a microplastic extraction protocol for examining green turtle (Chelonia mydas) chyme, which is multifarious in nature, by modifying and combining pre-established methods used to separate microplastics from organic matter and sediments. This protocol consists of visual inspection, nitric acid digestion, emulsification of residual fat, density separation, and chemical identification by Fourier transform infrared spectroscopy. This protocol enables the extraction of polyethylene, high-density polyethylene, (aminoethyl) polystyrene, polypropylene, and polyvinyl chloride microplastics >100µm. Two macroplastics and seven microplastics (two plastic paint chips and five synthetic fabric particles) were isolated from subsamples of two green turtles. Our results highlight the need for more research towards understanding the impact of microplastics on these threatened marine reptiles.