Cell-Derived Microparticles

Briefly, the two key features of NASH, steatosis and inflammation, were categorized as follows: steatosis was determined by analyzing hepatocellular vesicular steatosis, i.e. macrovesicular steatosis and microvesicular steatosis separately, and by hepatocellular hypertrophy as defined below (Fig. 2). Inflammation was scored by analyzing the amount of inflammatory cell aggregates (Fig. 2). The proposed rodent scoring system is shown in Table 4 and options for its use in diagnosis are shown in S1 Fig. The purpose of this scoring system is however not to derive a single score, but to score the individual features.
Macrovesicular steatosis and microvesicular steatosis were both separately scored and the severity was graded, based on the percentage of the total area affected, into the following categories: 0 (<5%), 1 (5–33%), 2 (34–66%) and 3 (>66%). The difference between macrovesicular and microvesicular steatosis was defined by whether the vacuoles displaced the nucleus to the side (macrovesicular) or not (microvesicular). Similarly, the level of hepatocellular hypertrophy, defined as cellular enlargement more than 1.5 times the normal hepatocyte diameter, was scored, based on the percentage of the total area affected, into the following categories: 0 (<5%), 1 (5–33%), 2 (34–66%) and 3 (>66%). For hepatocellular hypertrophy the evaluation was merely based on abnormal enlargement of the cells, irrespective of rounding of the cells and/or changes in cytoplasm or the number of vacuoles, and is therefore not a substitute of ballooning. The unweight sum of the scores for steatosis (macrovesicular steatosis, microvesicular steatosis and hypertrophy) thus ranged from 0–9. Both steatosis and hypertrophy were evaluated at a 40 to 100× magnification and only the sheets of hepatocytes were taken into account (terminal hepatic venules and portal tracts etc were excluded).
Inflammation was evaluated by counting the number of inflammatory foci per field using a 100 x magnification (view size of 3.1 mm²). A focus was defined a cluster, not a row, of ≥5 inflammatory cells. Five different fields were counted and the average was subsequently scored into the following categories: normal (<0.5 foci), slight (0.5–1.0 foci), moderate (1.0–2.0 foci), severe (>2.0 foci).
Hepatic fibrosis was identified using Sirius Red stained slides at 40 x magnification and evaluated by scoring whether pathologic collagen staining was absent (only in vessels) or collagen staining observed within the liver slide, the latter further defined as mild, moderate or massive. In addition, the percentage of the total area affected was evaluated using using image analysis of surface area on Sirius red stained slides.

To validate the scoring system, 36 slides of mouse livers covering the whole spectrum of NAFLD, were blindly analyzed by a board-certified pathologist (A.L.M), a clinical pathologist (A.D.) and nine scientists with basic histological experience. For the validation, the observers estimated the percentage of macrovesicular steatosis, microvesicular steatosis and hypertrophy (relative scale) and the number of inflammatory foci per field (absolute scale), instead of using the different categories for steatosis and inflammation (ordinal measure). Additionally, quantification of the steatosis and inflammation was determined by one observer during two separate assessments that were separated by an interval longer than 3 months.

For preparation of K₂CO₃ aqueous electrolytes, puratronic grade potassium carbonate salt
of 99.997% purity (metals basis) from Alfa Aesar was mixed with HPLC
LC-MS grade ultrapure water from VWR. For saturation with CO₂, the K₂CO₃ aqueous electrolytes were purged
with the gas for ∼30 min. A Si wafer (n-type doping, electric
resistivity ∼15 ± 3 MΩ cm) with a 50 nm Au thin
film on top of a 70 nm Ti film was obtained from MicroFabSolutions;
the wafer chip size was ∼12 × 12 mm². A polished
Au polycrystal of 1.7 mm thickness was supplied by Surface Preparation
Laboratory. A monodisperse 757 ± 19 nm polystyrene sphere suspension
of 5%/wt was obtained from MicroParticles GmbH.

Part of the serum obtained from the two groups of the study was used to quantify the level of Anti-S1RBD IgG antibodies. The quantification was performed using the ABBOTT SARS-CoV-2 IgG II Quant assay (REF# 6S60-22) on an ABBOTT ARCHITECT i1000SR instrument. The assay is an automated, two-step chemiluminescent microparticle immunoassay used for qualitative and quantitative determination of IgG antibodies against S1RBD of the SARS-CoV-2 from human serum and plasma. The SARS-CoV-2 IgG II Quant calibrator package (REF# 6S60-02) and the SARS-CoV-2 IgG II Quant control package (REF# 6S60-12) were run on the instrument prior to sample analysis. According to the manufacturer, the cut-off is set at 50.0 AU/mL, and the analytical measuring interval is set between 21.0 (limit of quantification) and 40,000.0 AU/mL (upper limit of quantification). Additional information on performance characteristics of the assay can be found in the manufacturer's manual. Based on the recommendations of the National Institute of Biological Standards and Control (NIBSC) and WHO, the concentrations were converted into Binding antibody units per mL (BAU/mL) through multiplying AU/mL by a factor of 0.142. The corresponding cut-off value becomes 7.1 BAU/mL.