Validation of morphometric analyses of small-intestinal biopsy readouts in celiac disease

Abstract

Background: Assessment of the gluten-induced small-intestinal mucosal injury remains the cornerstone of celiac disease diagnosis. Usually the injury is evaluated using grouped classifications (e.g. Marsh groups), but this is often too imprecise and ignores minor but significant changes in the mucosa. Consequently, there is a need for validated continuous variables in everyday practice and in academic and pharmacological research.

Methods: We studied the performance of our standard operating procedure (SOP) on 93 selected biopsy specimens from adult celiac disease patients and non-celiac disease controls. The specimens, which comprised different grades of gluten-induced mucosal injury, were evaluated by morphometric measurements. Specimens with tangential cutting resulting from poorly oriented biopsies were included. Two accredited evaluators performed the measurements in blinded fashion. The intraobserver and interobserver variations for villus height and crypt depth ratio (VH:CrD) and densities of intraepithelial lymphocytes (IELs) were analyzed by the Bland-Altman method and intraclass correlation.

Results: Unevaluable biopsies according to our SOP were correctly identified. The intraobserver analysis of VH:CrD showed a mean difference of 0.087 with limits of agreement from -0.398 to 0.224; the standard deviation (SD) was 0.159. The mean difference in interobserver analysis was 0.070, limits of agreement -0.516 to 0.375, and SD 0.227. The intraclass correlation coefficient in intraobserver variation was 0.983 and that in interobserver variation 0.978. CD3(+) IEL density countings in the paraffin-embedded and frozen biopsies showed SDs of 17.1% and 16.5%; the intraclass correlation coefficients were 0.961 and 0.956, respectively.

Conclusions: Using our SOP, quantitative, reliable and reproducible morphometric results can be obtained on duodenal biopsy specimens with different grades of gluten-induced injury. Clinically significant changes were defined according to the error margins (2SD) of the analyses in VH:CrD as 0.4 and in CD3(+)-stained IELs as 30%.

Figure 1. Graphical illustrations of the reliability and reproducibility of villus height crypt depth ratio.

Panels A and B show the Bland-Altman plots of small-intestinal mucosal villus height crypt depth ratio and Panels C and D present the regression analyses for intraobserver and interobserver analyses, respectively. The solid lines in panels A and B indicate the mean difference between the measurements and the dashed lines correspond to the 95% limits of agreement.

Figure 2. Two small-intestinal biopsy samples from routine clinics that show the importance of biopsy orientation in the interpretation of specimens.

Sectionings A and C are cut tangential and B and D perpendicular to the luminal surface showing the effect of different orientation to the same small-intestinal mucosal biopsy block. The hallmark of tangential cutting is the cross-sectioning of the crypts while in correct vertical cutting the crypts are cut longitudinally. In routine clinics, the tangentially cut sectioning A was interpreted as normal, and on re-evaluation upon high clinical suspicion of celiac disease, the biopsy block was tilted and recut. The recut biopsy sample (B) reveals crypt hyperplasia and villous atrophy compatible with celiac disease. To further highlight this potential source of diagnostic error, five independent pathologists were asked to interpret another biopsy block with slices cut in different planes. All graded the tangential specimen C to be morphologically normal (Marsh 0–1) and the properly oriented specimen D to have villus atrophy and crypt hyperplasia (Marsh 3b or 3c).

Figure 3. The intraepithelial lymphocyte densities are unaffected by the orientation of biopsy blocks.

Panels A and C show tangential (A) and perpendicular (C) cuttings of paraffin embedded blocks stained for CD3⁺ T cells, i.e. the same biopsy block as in Figures 2C and 2D. The panels B and D present 40× magnified pictures of the mucosa from the places presented by the black rectangles in panels A and C. In these magnifications, the intraepithelial lymphocyte densities are 50 per 100 epithelial cells and 54 per 100 epithelial cells, respectively.

Figure 4. Computerized 3D-model demonstrates the effects of correct and incorrect planes of cutting on readout results.

In the middle column are the biopsy blocks, in which the dashed and solid lines represent planes of sectioning. In the left column are sectionings cut perpendicular to the luminal surface and in the right tangentially cut sectionings. For example, in the middle row the computerized block shows merged and convoluted low villous ridges which in perpendicular cutting results in subtotal villous atrophy with deep crypts (left) but in tangential cutting in tall villi with only cross-sections of crypts (right).