Xn 10

Routine tests were performed on 30 μl of mouse blood following the manufacturer’s instructions (Sysmex XN-10) for dilution mode.

Blood samples were collected from the mock circuits into different blood collection tubes directly before and after six hours blood perfusion of the endothelialized and non-endothelialized HFMs. Blood was collected in citrate tubes (1:10) 2.9 mL (Sarstedt, Nürmbrecht, Germany) for D-Dimer measurement using turbidimetry on the Atellica COAG 360 (Siemens, München, Germany). Determination of the thrombocyte count was carried out with blood collected in K3 EDTA 1.6 mL tubes (Sarstedt, Nürmbrecht, Germany), using impedance measurement on the XN-10 (Sysmex, Kobe, Japan). The relative change in D-Dimer concentrations and thrombocyte counts was expressed in percentages, which were calculated by subtracting the values obtained after flow exposure from the corresponding baseline values before flow divided by the baseline values and multiplied by 100%.

The laboratory assessment was performed after an overnight fast and included liver biochemistry (alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, gamma-glutamiltransferase (GGT), alkaline phosphatase (AP) and albumin) and metabolic parameters (lipid profile, uric acid and fasting serum insulin and glucose). All tests were performed at the Central Laboratory of Hospital das Clínicas, Universidade Federal de Minas Gerais. Plasma glucose levels, bilirubin, albumin, triglycerides, and total cholesterol and fractions were quantified by colorimetric/dry chemistry assay (VITROS^® 5600 Integrated System, Hong Kong, China). Insulin was measured by chemiluminescence immunoassay (Architect I1000SR^®, Wiesbaden, Germany) and IR was defined by HOMA values >3 [31 (link)]. ALT, AST, AP and GGT were measured by enzymatic kinetic/dry chemistry assay (VITROS^® 5600 Integrated System, Hong Kong, China). Platelet count was determined by an automated hematology analyzer (Sysmex XN10, Chuo-ku, Japan).
NAFLD fibrosis score [32 (link)] and all the other laboratory evaluations were determined at baseline and after the treatment (synbiotic group) or three months after the first tests (control group).

Complete blood counts were performed on a XE-5000 or XN-10 device from Sysmex (Norderstedt, Germany) in order to determine leukocyte and thrombocyte counts. Patient serum was obtained using Serum-Gel S-Monovettes (Sarstedt, Nümbrecht, Germany), aliquoted, and stored at −80 °C until further analysis. C-reactive protein was measured on the c702 turbidimetry module of the cobas 8000 analyzer from Roche (Mannheim, Germany). Complement factors C3 and C4 were also measured with turbidimetric assays on the c502 module as well on the cobas 8000 device. IgG, IgG2, IgG4, and IgE were quantified on the BN Prospec nephelometer from Siemens (Eschborn, Germany).

Glucose was measured using Cobas 6000 (Roche Diagnostics) or with a spectrophotometric method using Radiometer ABL 800 flex Blood Gas Analyzer which uses the hexokinase method on serum. Hemoglobin (Hb) was measured with a spectrophotometry method using Radiometer ABL 800 flex Blood Gas Analyzer or using the Sysmex XN-10, using a spectrophotometric method on hemolyzed blood. Creatinine was analyzed using Cobas 6000 (Roche Diagnostics) or with a spectrophotometric method using Radiometer ABL 800 flex Blood Gas Analyzer. Details on analytical and reference ranges for these analyses can be found in the Additional file 1.
Samples of hs-cTnT were collected in lithium heparin tubes and analyzed with the Roche Cobas e602 (Roche Diagnostics). This assay has a limit of detection of 5 ng/L and a limit of blank of 3 ng/L. Coefficient of variation is < 10% at 13 ng/L and the 99^th percentile cut-off point is at 14 ng/L [21 (link)].