Refractive index holotomography for quantifying nuclear features in clear cell renal cell carcinoma WHO/ISUP grading

Clear cell renal cell carcinoma (ccRCC) is classified by the four-tiered World Health Organization/International Society of Urological Pathology (WHO/ISUP) grading system based on nucleolar prominence (grades 1-3) and nuclear anaplasia (grade 4) after Hematoxylin and Eosin staining. However, manual evaluation often leads to poor inter- and intra-observer reproducibility. We applied holotomography, a label-free imaging technique for measuring the refractive index (RI) of biological specimens, to quantify the RI differences in segmented nuclei by grade. The RI distribution within all nuclei and RI standard deviations in local patches significantly differed by grade (Student’s t-test, p<0.001). Grades 1 through 3 exhibited a decreasing trend, whereas grade 4 deviated from this pattern, reflecting the WHO/ISUP grading criteria. Morphology, intensity, and texture-related RI features also varied significantly among the grades (p<0.001). This quantitative approach to ccRCC grading can reduce reliance on subjective evaluation, thereby improving diagnostic accuracy.