2.4. Quantitative Analysis of Optical Coherence Tomography and Histology Data

Following the RFA application and OCT imaging, the specimens used in both protocols were inked to indicate the OCT imaged area, placed in standard tissue cassettes, and fixed in 10% formalin prior to histology processing. Standard hematoxylin and eosin (H&E) staining was used to assess the coagulum from the RFA application.^13,14 Digital pathology images were captured from the H&E histology slides by using a slide scanner (Aperio AT2, Leica Biosystems, Illinois) with a 20× magnification. Both the OCT images and corresponding H&E histology images were analyzed to identify the coagulum characteristics, including the coagulum thickness.

Post-RFA OCT images were examined in order to segment the coagulum, residual EP, and underlying lamina propria (LP) layers manually, based on the difference in scattering properties. The thickness of the coagulum and coagulum + residual EP layer was measured in individual cross-sectional OCT images by assuming a tissue refractive index of 1.38 at ∼1310-nm wavelength.³¹ A 2-D thickness map was generated from volumetric post-RFA OCT datasets. The average, as well as minimum and maximum thickness, was obtained from the OCT measurements of individual specimens (volumetric or single cross-sectional OCT images) in order to assess the effects of RFA. For the subset of specimens processed for histology, ∼4 to 5 histological sections with a step interval of 200μm were obtained from the OCT imaged region. The thickness of the coagulum and residual EP layer was manually measured at multiple locations using a digital pathology viewer (Aperio ImageScope v.11, Leica Biosystems, Illinois). Similar to the OCT measurement, the average, as well as minimum and maximum thickness, was measured and compared with the corresponding OCT measurement.