Digital image analysis of rhodanine-stained liver biopsy specimens for calculation of hepatic copper concentrations in dogs

Objective—To evaluate the accuracy of digitally scanned rhodanine-stained liver biopsy specimens for determination of hepatic copper concentration and compare results with qualitatively assigned histologic copper scores in dogs. Sample—353 liver biopsy specimens from dogs. Procedures—Specimens (n = 139) with quantified copper concentration ranging from 93 to 6,900 μg/g were allocated to group 1 (< 400 μg/g [37]), group 2 (401 to 1,000 μg/g [27]), group 3 (1,001 to 2,000 μg/g [34]), and group 4 (> 2,001 μg/g [41]); stained with rhodanine; and digitally scanned and analyzed with a proprietary positive pixel algorithm. Measured versus calculated copper concentrations were compared, and limits of agreement determined. Influence of nodular remodeling, fibrosis, or parenchymal loss on copper concentration was determined by digitally analyzing selected regions in 17 specimens. After method validation, 214 additional liver specimens underwent digital scanning for copper concentration determination. All sections (n = 353) were then independently scored by 2 naive evaluators with a qualitative scoring schema. Agreement between assigned scores and between assigned scores and tissue copper concentrations was determined. Results—Linear regression was used to develop a formula for calculating hepatic copper concentration ≥ 400 μg/g from scanned sections. Copper concentrations in unremodeled specimens were significantly higher than in remodeled specimens. Qualitative scores widely overlapped among quantitative copper concentration groups. Conclusions and Clinical Relevance—Calculated copper concentrations determined by means of digital scanning of rhodanine-stained liver sections were highly correlated with measured values and more accurate than qualitative copper scores, which should improve diagnostic usefulness of hepatic copper concentrations and assessments in sequential biopsy specimens.