Stereological Analysis of Liver Biopsy Histology Sections as a Reference Standard for Validating Non-Invasive Liver Fat Fraction Measurements by MRI

Abstract

Background and aims: Validation of non-invasive methods of liver fat quantification requires a reference standard. However, using standard histopathology assessment of liver biopsies is problematical because of poor repeatability. We aimed to assess a stereological method of measuring volumetric liver fat fraction (VLFF) in liver biopsies and to use the method to validate a magnetic resonance imaging method for measurement of VLFF.

Methods: VLFFs were measured in 59 subjects (1) by three independent analysts using a stereological point counting technique combined with the Delesse principle on liver biopsy histological sections and (2) by three independent analysts using the HepaFat-Scan® technique on magnetic resonance images of the liver. Bland Altman statistics and intraclass correlation (IC) were used to assess the repeatability of each method and the bias between the methods of liver fat fraction measurement.

Results: Inter-analyst repeatability coefficients for the stereology and HepaFat-Scan® methods were 8.2 (95% CI 7.7-8.8)% and 2.4 (95% CI 2.2-2.5)% VLFF respectively. IC coefficients were 0.86 (95% CI 0.69-0.93) and 0.990 (95% CI 0.985-0.994) respectively. Small biases (≤3.4%) were observable between two pairs of analysts using stereology while no significant biases were observable between any of the three pairs of analysts using HepaFat-Scan®. A bias of 1.4±0.5% VLFF was observed between the HepaFat-Scan® method and the stereological method.

Conclusions: Repeatability of the stereological method is superior to the previously reported performance of assessment of hepatic steatosis by histopathologists and is a suitable reference standard for validating non-invasive methods of measurement of VLFF.

Fig 1. Stereological analysis of biopsy sections.

(a) Example histological section of a liver biopsy with square grid randomly placed over image. The grid size was adjusted so that approximately 200 intersections are located within the tissue boundary. Every intersection within the tissue boundary is visually examined to determine whether or not it falls on a fat vesicle. The volumetric fat fraction for this example was determined to be 18.6%. (b) High magnification image of four of the intersections in (a). The lower left grid intersection was assessed to be within a fat vesicle while the other three intersections were assessed to be outside fat vesicles. The distance between two neighbouring intersections is 250 microns or 504 pixels in this example. Note that the grid lines are shown thicker here than in the analysis for clarity of display. During analysis the grid lines are one pixel wide at the highest magnification.

Fig 2. Magnetic resonance images of the liver.

(a) A typical region of interest selected for analysis within the liver magnetic resonance image. (b) A typical region of interest of free space selected within the MR image for measurement of background noise.

Fig 3. Inter-analyst comparison of the stereology results.

Comparisons of results of measurement of volumetric liver fat fraction in 59 biopsy sections by three independent analysts (identified as S, M, and W) using the stereology method. The left column shows plots of the results from one analyst against the results from another. The solid line is the line of equivalence (not a line of best fit). The right columns shows Bland Altman plots of the difference in results between two analysts plotted against the mean result from two analysts. The horizontal solid line indicates the mean difference between the two analysts while the dashed lines indicate the upper and lower 95% limits of agreement between the two analysts.

Fig 4. Inter-analyst comparison of the HepaFat-Scan^® MRI results.

Comparisons of results of measurement of volumetric liver fat fraction in 59 MRI datasets by three independent analysts (identified as R, S, and W) using the HepaFat-Scan^® method. The left column shows plots of the results from one analyst against the results from another. The solid line is the line of equivalence (not a line of best fit). The right columns shows Bland Altman plots of the difference in results between two analysts plotted against the mean result from two analysts. The horizontal solid line indicates the mean difference between the two analysts while the dashed lines indicate the upper and lower 95% limits of agreement between the two analysts.

Fig 5. Inter-analyst comparison of hepatopathologists results.

Comparisons of results of assessment of percentage steatosis in 59 biopsy sections by three independent hepatopathologists (identified as AC, BD, and PB). The left column shows plots of the results from one hepatopathologist against the results from another. The solid line is the line of equivalence (not a line of best fit). The right columns shows Bland Altman plots of the difference in results between two hepatopathologists plotted against the mean result from two hepatopathologists. The horizontal solid line indicates the mean difference between the two hepatopathologists while the dashed lines indicate the upper and lower 95% limits of agreement between the two hepatopathologists.

Fig 6. Comparison of volumetric liver fat fraction measured by HepaFat-Scan^® and stereology.

(a) Volumetric liver fat fraction measured by HepaFat-Scan^® versus the volumetric liver fat fraction measured by stereology from histology sections of liver biopsy samples. The solid line is the line of equivalence (not a line of best fit). (b) Bland Altman plot showing the differences of the volumetric liver fat fractions measured by HepaFat-Scan^® and by stereological analysis of liver biopsy histology sections plotted against mean of the two measurements. The solid line indicates the mean difference while the dashed lines indicate the upper and lower 95% limits of agreement between the two measurements.