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.
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.
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