Computational protocol: Whole slide image with image analysis of atypical bile duct brushing: Quantitative features predictive of malignancy

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Protocol publication

[…] Over a 3-year consecutive period, the pathology database was searched for BD brush specimens with indeterminate diagnostic categorization. These included “atypical” or “suspicious” in the cytopathologic report during the course of the routine patient evaluation. Cases reported as benign, and adenocarcinoma/malignant/positive were excluded. For this indeterminate group, the corresponding pathology database was cross-referenced, and specimens with a corresponding histologic confirmation of carcinoma were selected. Only cases with an indeterminate cytopathologic diagnosis (atypical/suspicious) and histologic diagnosis of carcinoma were included in the study group. Pathology reports were collected and patient demographics and data were reviewed. BD brush specimens are processed from a liquid fixative (PreservCyt™, Hologic Corporation) and a single non-Gyn Filter ThinPrep™ slide (Hologic Corporation) is made. It is stained by a traditional Papanicolaou staining method and interpreted by a pathologist. The individual slides from each individual case were placed in an Aperio® Scanscope XT 120 (Leica Corporation) slide scanner and using the ×20 (doubler) scanning process, a WSI was created.Within the Aperio® Spectrum™ software, an individual case was opened in the ScanScope/ImageScope™ software (v10.2.2.2352 Leica Biosystems Inc., Buffalo Grove, Illinois, USA). The entire WSI was available for review. Using the scroll and zoom in/out capability, the entire slide was systematically reviewed by a single operator without knowledge of original categorization or patient outcome. Postprocessing image adjustment built into the software (brightness/contrast) was adjusted to improve visualization of three-dimensional groups and better identify individual nuclear outlines within a group. The operator was instructed to identify the most cytologically abnormal groups that could be well-visualized and the 10 best were manually selected. These were identified and the outer limit of each individual group was delineated using the “pen tool” which is available in the ImageScope™ software. The operator identified the outer limits of the group cytoplasm and completely encircled the entire group. Groups with benign features such as small orderly monomorphic nuclei and flat sheets were not selected. Instances where the groups were difficult to visualize, or other characteristics were not clearly apparent were not selected; the operator used their best judgment based on the individual group's morphology. Exclusion criteria included single epithelial cells, groups of indeterminate origin (secondary to crush, poor preservation, or staining), and nonepithelial cells (neutrophils, singly, and in clusters). Ten groups were selected and then a manual process was used to delineate the outside margins of the group and individual nuclei []. Within the 10 selected individual groups, the individual nuclei were manually selected and delineated by the “pen tool” []. These qualified and delineated “pen tool” groups were recorded within the ImageScope™ software in the “annotations” dialog box. Within the “annotations” dialog box, each individual group is given a “region” designation under a single “layer” category []. Ten of these were recorded for each analyzed group in individual cases. At the completion of the process, the individual region designation allowed the operator to review each group by clicking on the “region” which then takes the view screen to a high power encompassing view of the specific group. The entire manual process of group and nuclei selection took an average of 15–20 min/WSI. After completing the process of delineating groups of interest for the WSI, the objective quantitative image analysis process was performed. The manual process of delineating the groups of interest was performed by a single operator (RCW).Each individual group was quantitatively analyzed for the number of total nuclei, individual nuclear area (µm2), and total group area (µm2). Based on the known magnification of the images by the software program, these are calculated directly. From this directly measured data, the average number of nuclei per group, average nuclear to cytoplasmic (N/C) ratio per group and range of nuclear area (nuclear size differential) per group were calculated.After the analysis, the cumulative WSI “layer” region result is exported by the ImageScope™ software to a Microsoft Excel™ spreadsheet file which has an individual line for each delineated and measured region. A large amount of data is recorded, only some of which is pertinent and utilized. The directly measured data fields used are: Group area (µm2), total nuclei per group (numeric), and individual nuclear size (µm2) []. From the Excel™ spreadsheet, the measured group size is known, and the total nuclear area calculated (total nuclei area added per group), and then the (N/C) ratio is calculated by dividing the measured total nuclear area by the measured group area (nuclear area/group area). The overall calculated average N/C ratio for the individual WSI is obtained by taking the added total of each N/C ratio and dividing it by the total group number (total of all N/C ratio/total group number). The range or difference in nuclear size per each individual group was measured, and then the average nuclear size differential per WSI was calculated.Once the calculations were completed independently and in a blinded fashion, the individual cases were correlated with the pathologic reports. The BD brush WSI cases were categorized into “atypical” and “suspicious” groups based on the original pathologic classification. Data analysis on the Microsoft® Excel file was performed by the Excel™ software program on individual cases and for analysis of group categories and associated measured outcomes. The P values were calculated using standard calculation formulas and independent two sample t-test calculations (IBM Corporation, SPSS Statistics, version 21 Armonk, New York. USA). A result was considered statistically significant if P < 0.05. A relative risk ratio was calculated, and it was intended to provide information about the image analysis cut-off values. It is not a standard risk ratio of malignancy. The risk ratio is calculated utilizing the cases that were equal to or greater than the cut-off values as a true positive result. This is intended to help stratify the image analysis features within the context of these indeterminate cytopathology brush classifications where patients were determined to have carcinoma. The study had the approval of the Institutional Review Board. […]

Pipeline specifications

Software tools ImageScope, SPSS
Applications Miscellaneous, Whole slide imaging
Diseases Bile Duct Diseases, Carcinoma, Neoplasms