Double staining with crystal violet and methylene blue is appropriate for colonic endocytoscopy: An in vivo prospective pilot study

Abstract

Background and AimEndocytoscopy (EC) at ultra-high magnification enables in vivo visualization of cellular atypia of gastrointestinal mucosae. Clear images are essential for precise diagnosis by EC. The aim of the present study was to evaluate the optimal staining method for EC in the colon.MethodsThirty prospectively enrolled patients were allocated 1:1:1 to three distinct staining methods: 0.05% crystal violet (CV) alone, 1% methylene blue (MB) alone, or CV + MB (CM double). Normal rectal mucosae were stained with each dye and videos of EC images were recorded. Visibility of nuclei and gland formation after staining were evaluated as ‘recognizable’ or ‘not recognizable’. Time for each parameter to become ‘recognizable’ was measured, and the average times for the three staining regimens were compared.ResultsMB alone and CM double staining resulted in ‘recognizable’ (102 ± 27 vs 89 ± 22 s, P = 0.263) nuclei within comparable periods of time, whereas CV alone was unable to identify nuclei. Gland formation became ‘recognizable’ sooner after CM double staining than after MB alone (61 ± 16 vs 108 ± 24 s, P < 0.001).ConclusionsDouble staining with CV and MB, which rapidly provided recognizable images of both nuclei and gland formation, is an appropriate staining regimen for colonic EC.

Evaluation of microvascular findings of deeply invasive colorectal cancer by endocytoscopy with narrow-band imaging

“…5). This may have been attributable to increased light intensity, increased exposure time of NBI, and improved noise reduction, thereby allowing clearer observation of the vessels and detailed examination of EC-V. With regard to advantages of EC-NBI over conventional EC, conventional EC always requires staining with crystal violet and methylene blue 16 17. However, EC-NBI does not require staining, and testing with EC-NBI therefore can be performed safely and quickly.…”

Endoscopic Imaging

“…Studies in esophagus [46], stomach [47], and colon [48•] provide in vivo histology to identify histological characteristics. The combination of two contrast agents methylene blue and crystal violet allows for improved characterization of both epithelial structures and nuclei [49].…”

Advanced colonoscopic imaging using endocytoscopy

“…Recently, a new staining approach was introduced by Ichimasa and coworkers . The new approach consists of double staining containing crystal violet and methylene blue.…”

“…Those differences were calculated as statistically significant. It was therefore concluded that double staining with CV and MB, which rapidly provided recognizable images of both nuclei and gland formation, is an appropriate staining regimen for colonic endocytoscopic imaging …”