HE-referenced eosinophil assessment and cutoff estimation using Congo red and Chromotrope 2R staining in chronic rhinosinusitis with nasal polyps

This cross-sectional histopathological study aimed to compare eosinophil visualization and quantification using Congo red, Chromotrope 2R, and conventional hematoxylin–eosin (HE) staining in chronic rhinosinusitis with nasal polyps (CRSwNP), and to explore HE-referenced eosinophil-count cutoff estimates for Congo red and Chromotrope 2R staining in relation to conventional HE-based classification of eosinophilic CRSwNP (eCRSwNP). Three consecutive 4 μm sections of 103 paraffin-embedded nasal polyps were stained with Congo red, Chromotrope 2R, and HE, respectively. For each case, five corresponding nonoverlapping high-power fields were selected from serial sections for eosinophil assessment. Eosinophils were quantitatively counted and compared among the three staining methods. Using conventional HE-based classification as the routine histopathological comparator, receiver operating characteristic (ROC) curve analysis was performed to explore HE-referenced eosinophil-count cutoff estimates for Congo red and Chromotrope 2R staining, with optimal cutoff estimates determined by the Youden index. Congo red and Chromotrope 2R provided clearer eosinophil visualization than conventional HE staining. Eosinophil counts differed significantly among the three staining methods (P < 0.001). Using conventional HE-based classification as the routine histopathological comparator, the areas under the receiver operating characteristic curves (AUCs) for Congo red and Chromotrope 2R staining were 0.9967 (95% CI, 0.990–1.000) and 0.9972 (95% CI, 0.992–1.000), respectively. The Youden index-derived HE-referenced cutoff estimate was ≥ 14.3 eosinophils/HPF for Congo red staining, with a sensitivity of 100% (95% CI, 94.22%-100%) and a specificity of 95.12% (95% CI, 83.47%-99.40%). For Chromotrope 2R staining, the Youden index-derived HE-referenced cutoff estimate was ≥ 14.6 eosinophils/HPF, with a sensitivity of 95.16% (95% CI, 86.50%-98.99%) and a specificity of 100% (95% CI, 91.40%-100%). Congo red and Chromotrope 2R staining may facilitate eosinophil assessment and provide histopathological support for HE-referenced eCRSwNP classification, particularly when eosinophil identification on HE-stained sections is difficult.