Morphometric and visual evaluation of fibrosis in renal biopsies

Abstract

Interstitial fibrosis is an outcome measure of increasing importance in clinical trials of both renal transplantation and native disease, but data on the comparative advantages of fibrosis measurement methods are limited. We compared four morphometric techniques and contrasted these with two visual fibrosis-scoring methods on trichrome-stained slides. Two morphometric methods included whole-slide digital images: collagen III immunohistochemistry and a new technique using trichrome and periodic acid-Schiff subtraction morphometry; the other two methods included Sirius Red with and without polarization on multiple digital fields. We evaluated 10 serial sections from 15 renal biopsies with a range of fibrosis extent and diagnoses on duplicate sections with each method on separate days. Three pathologists performed visual scoring on whole-slide images. Visual and morphometric techniques had good to excellent interassay reproducibility (R(2) = 0.62 to 0.96) and interobserver reproducibility (R(2) = 0.75 to 0.99, all P < 0.001). Morphometry showed less variation between observers than visual assessment (mean of 1% to 5% versus 11% to 13%). Collagen III, Sirius Red unpolarized, and visual scores had the strongest correlations (R(2) = 0.78 to 0.89), the greatest dynamic range, and the best correlation with estimated GFR (R(2) = 0.38 to 0.50, P < 0.01 to 0.001). Considering efficiency, reproducibility, and functional correlation, two current techniques stand out as potentially the best for clinical trials: collagen III morphometry and visual assessment of trichrome-stained slides.

Figure 1.

The study involved serial sections with staining performed on different days, denoted day 1 and day 2.

Figure 2.

Quantitation of fibrosis using Trichrome-PAS staining. (A) Trichrome and PAS stains. (B) The corresponding “mark-up” image generated by the quantitation algorithm applied to the Trichrome and PAS stains. Tissue considered “positive” is “marked up” either yellow, orange, or red, in that order, with increasing positivity of match to the algorithm parameters. Note that basement membranes, sclerotic glomeruli, and blood vessels stain with both the trichrome and PAS stains and are thus “subtracted” with the PAS stain. Tubular brush borders were not falsely detected. Casts stained with PAS stains were thus subtracted. (C) Graph of trichrome, PAS, and T-P fibrosis values for both days combined, arranged by increasing trichrome-PAS area.

Figure 3.

Quantitation of fibrosis using collagen III immunohistochemistry. (A) Collagen III immunohistochemistry stain. (B) The corresponding “mark-up” image generated by the quantitation algorithm applied to the collagen III immunohistochemistry stain. Tissue considered “positive” is “marked up” either yellow, orange, or red, in that order, with increasing positivity of match to the algorithm parameters.

Figure 4.

Quantitation of fibrosis using Sirius Red staining. (A) Under polarized light. (B) Screen shot of the quantitative analysis performed on the Sirius Red stain.

Figure 5.

Visual fibrosis quantitation methods. (A) The first method involved the human simulating how the computer would assess fibrosis. The fibrosis percentage was taken as the percentage of all tissue (excluding tubules and glomeruli) occupied by fibrous tissue. (B) The second method involved assessing the percentage of the tissue that is abnormal (or, conversely, 100 − % normal).

Figure 6.

Correlation of visual assessments. Regression lines of pathologist's visual assessments and R² values of corresponding measurements show how measurements correlate with day 1 and day 2 sections of each sample averaged. For all P < 0.0001. Curved lines bound a density ellipse containing 95% of the measurements obtained. Regressions are shown for each pathologist (numbered observer 1, 2, and 3) for each visual assessment method (% fibrous and % abnormal).

Figure 7.

Correlation of computer-based and visual measurements. Regression lines show the correlations of computer-based and visual measurements with measurements of day 1 and day 2 sections of each sample averaged. Regression R² values are given for the corresponding combined day 1 and day 2 measurements. Curved lines bound a density ellipse containing 95% of the measurements obtained. Corresponding P values are given in Table 3. SR, Sirius Red; T-P; trichrome-PAS.

Figure 8.

Correlation of morphometry and renal function. This example of a regression of % fibrosis by collagen III with a clinical parameter, the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) estimated GFR (eGFR), shows that there is a moderate correlation between morphometry and renal function. Additional correlation coefficients (R²) are shown in Table 5.