The use of Masson's trichrome staining, second harmonic imaging and two‐photon excited fluorescence of collagen in distinguishing intestinal tuberculosis from Crohn's disease

AIM: Differentiation between Crohn's disease (CD) and intestinal tuberculosis (ITB) continues to be difficult. The present study investigated the collagen fibre characteristics of CD and ITB using Masson's trichrome staining, second harmonic generation (SHG) imaging and two-photon excited fluorescence (TPEF) imaging with the aim of distinguishing between them. METHOD: The characteristics of collagen fibres in intestinal specimens from patients with CD, ITB and healthy controls were compared using Masson's trichrome staining and SHG and TPEF imaging. RESULTS: Masson's trichrome staining showed that the content of collagen fibre (540.92 [139.61-1681.93] vs 236.17 [72.94-1108.32], P < 0.05) and fibre deposits (888.92 [315.89-3172.9] vs 498.98 [38.82-5802.31], P < 0.05) were both higher in ITB than in CD. The content of collagen fibre (594.677 [139.61-1681.93] vs 107.425 [4.66-988.7], P < 0.05) and fibre deposits (1118.4661 [315.89-5802.31] vs 340.575 [29.62-1188.87], P < 0.05) were significantly higher in lesions with granulomata than in those without. The SHG/TPEF images demonstrated that the percentage of fibrosis in ITB was also significantly higher than in CD (P < 0.05), in both surgical (13.363% ± 5.303% vs 8.322% ± 5.078%, P = 0.044) and endoscopic specimens (mean rank 13.5 vs 7.5, P = 0.023). The SHG/TPEF imaging described different distribution patterns of collagen between CD and ITB; in the former this was irregular in clumps while in ITB the collagen was arranged around caseating granulomata. CONCLUSION: The evaluation of fibrosis in CD and ITB by Masson's trichrome staining and SHG and TPEF imaging appears to distinguish between these two diseases.