An Innovative Transmission Electron Microscopy-Based Ultrastructural Imaging Methodology for the Diagnosis of Respiratory Ciliary Disorders.

Abstract

Transmission electron microscopy is a classical methodology for diagnosing many ciliary disorders. However, heterogeneity in sampling, low yield of ciliary epithelium by cytology, improper fixation, and different orientations of cylindrical axes of cilia section create difficulties in achieving the appropriate ciliary ultrastructure for diagnosis. We have reported an improvement in the number and proportion of ciliary epithelial cells up to 32 times. These include cleaning the nose before sample collection, changing the collection brush used for the cervical cytology (eight times), and paired nose collection (four times). We have optimized the primary fixation to preserve cilia ultrastructure, modified ultra-sectioning, and improved the number of reportable ultrastructure of cilia by tilting the sample angles of α axes (up to ±40°) and β axes (up to ±25°). These optimizations resulted in ∼20 times more reportable cilia (parallel to cylindrical axes concerning the electron beam, clearly visible dynein arms, 9 + 2 patterns, and compound cilia). This resulted in an enhanced diagnosis of ciliary disorders, up to 67% (reported in 135 among 200 patients), compared with 5% achieved using conventional routine practices at our facility. With certain advancements in conventional method, we could achieve an accuracy comparable to the new sophisticated cryo-electron tomography imaging.