Comparison Of Lymphatic Staining Methods And Partial Reconstruction Based On Whole Organ Sections Of Laryngeal Carcinoma

Objective:This study is performed to compare the diagnostic potential of HE,enzymatic histochemical and immunohistochemical determination of lymphatic vessel in laryngeal carcinoma.On this basis,the positive staining structure of the more reliable method was reconstructed to verify the accuracy of the relevant staining methods.Methods:We recruited three patients who were pathologically diagnosed as laryngeal squamous cell carcinoma and performed total laryngectomy.According to the improved Kawamoto's Film Method,frozen specimens of whole laryngeal squamous cell carcinoma were made,Immunohistochemistry,that is,using lymphatic endothelial cell specific marker D2-40,enzymatic histochemistry(5-nucleotidase)and HE staining were used to stain the frozen sections of laryngeal squamous cell carcinoma.The sections were stained on the adhesive film and preserved between the adhesive film and glass slide.Then,the staining results of lymphatic vessels in the specimens of laryngeal carcinoma were compared by observation under the optical microscope.Furthermore,the better single stained slices were sequentially registered,segmented and reconstructed.Results:The background of HE and D2-40 immunohistochemical staining were clear.5-Nase staining had a deep background and brown color.By immunohistochemistry,102(97.1%,102/105)blood vessels were identified,3(2.9%)were partly positive.While using 5-Nase,56(53.3%,56/105)blood vessels were identified.In 105 lumen structures that could not be clearly judged,95(90.5%)were positive by D2-40,while in 5-Nase staining,89(84.8%)were positive including a large number of glands.In addition,positive cells scattered in dots or clusters were observed in 5-Nase and D2-40 staining sections,and these cells or structures could not be identified in HE staining.After the whole registration of D2-40 staining sections,the region of interest was selected for three-dimensional reconstruction.It was found that the tubular structure with D2-40 staining was columnar distribution,while the dotted structure with D2-40 staining was clustered distribution.Conclusion:By using the improved Kawamoto's Film Method,the adverse effects caused by the enlargement of the size of the slices are overcome,and higher quality stained slices are obtained.Combining with the results of two-dimensional staining and three-dimensional reconstruction of positive staining structures of D2-40 sections,it is suggested that D2-40 has high specificity and accuracy for the labeling of lumen-like lymphatics in laryngeal cancer,while the accuracy of non-lumen-like lymphatics still needs further verification.