Purpose: We describe registration accuracy studies of a custom hardware-software system called eeDAP that registers fields of view (FOVs) of a glass slide on a microscope to the digital presentations of regions of interest (ROIs) of whole slide images (WSI) of the same glass slide. In this manuscript, we describe the results of adding new hardware and use the results to size a larger pathologist data collection study.
Methods: We create a registration accuracy task by identifying a visually distinct target. This target will be the center of a WSI ROI and is expected to appear in the center of the microscope FOV. We examined the registration accuracy of 60 ROIs from six slides, alternating registration methods and slide order within each study. We measure the distance between the target and the FOV center (registration error) using an eye piece reticle ruler as the stage moves from target to target. We summarize each error as a success (≤ 5.0 µm) or failure (> 5.0 µm). We completed a multi-reader multicase (MRMC) analysis of the registration successes and failures to estimate the variance components due to the readers and the cases.
Results: When using eeDAP in-focus, accuracy was within 5 µm in more than 97% of the FOVs.
Conclusions: The eeDAP registration methods were robust to new hardware, and the MRMC analysis has provided variance components for sizing future registration accuracy studies to account for the variability from readers and cases.
Purpose: Validation of artificial intelligence (AI) algorithms in digital pathology with a reference standard is necessary before widespread clinical use, but few examples focus on creating a reference standard based on pathologist annotations. This work assesses the results of a pilot study that collects density estimates of stromal tumor-infiltrating lymphocytes (sTILs) in breast cancer biopsy specimens. This work will inform the creation of a validation dataset for the evaluation of AI algorithms fit for a regulatory purpose.Approach: Collaborators and crowdsourced pathologists contributed glass slides, digital images, and annotations. Here, “annotations” refer to any marks, segmentations, measurements, or labels a pathologist adds to a report, image, region of interest (ROI), or biological feature. Pathologists estimated sTILs density in 640 ROIs from hematoxylin and eosin stained slides of 64 patients via two modalities: an optical light microscope and two digital image viewing platforms.Results: The pilot study generated 7373 sTILs density estimates from 29 pathologists. Analysis of annotations found the variability of density estimates per ROI increases with the mean; the root mean square differences were 4.46, 14.25, and 26.25 as the mean density ranged from 0% to 10%, 11% to 40%, and 41% to 100%, respectively. The pilot study informs three areas of improvement for future work: technical workflows, annotation platforms, and agreement analysis methods. Upgrades to the workflows and platforms will improve operability and increase annotation speed and consistency.Conclusions: Exploratory data analysis demonstrates the need to develop new statistical approaches for agreement. The pilot study dataset and analysis methods are publicly available to allow community feedback. The development and results of the validation dataset will be publicly available to serve as an instructive tool that can be replicated by developers and researchers.
Increased utilization of transplantation as treatment for patients with end-stage hepatic disease has resulted in a shortfall of available livers. Efforts to expand the available donor pool have resulted in the inclusion of donors who might not have been considered in the past. This has resulted in more requests for frozen section biopsy evaluation of the liver from "marginal" donors with significant co-morbidities. The information gained from the biopsy analysis determines whether the organ is suitable for transplantation. Critical to determining the adequacy of donor livers is analyzing the lipid content for macrosteatosis; high lipid livers are not suitable for transplant. Frozen section analysis (FSA) creates artifacts that limit tissue evaluation, exhausts tissue for downstream histological analysis, and requires a specialized team to evaluate these procedures in the hospital 24/7. We have developed a fluorescence microscopy system that utilizes structured illumination (SIM) to produce images of liver biopsies within seconds of removal from a deceased organ donor. Liver biopsies that require evaluation for donation suitability are stained with fast-acting fluorescent histology dyes and lipid specific stains in order to differentiate the lipids on SIM. The SIM images are compared to the standard-of-care FSA and the final pathology report. Here, we present the results of this blinded review performed by a liver pathology specialist. Imaging liver biopsies with SIM provides a more direct and accurate tool for determining macrosteatosis compared to standard FSA. SIM offers minimal tissue processing complexity and remote viewing capabilities, creating the potential to revolutionize tissue donation evaluation.
Currently available pathology techniques for obtaining a rapid tissue diagnosis, or for determining the adequacy of specimens intended for downstream analysis, are too slow, labor-intensive, and destructive for point-of-care (POC) applications. We previously demonstrated video-rate structured illumination microscopy (VR-SIM) for accurate, high-throughput, non-destructive diagnostic imaging of fluorescently-stained prostate biopsies in seconds per biopsy, with an area under the ROC curve of 0.82-0.88 after pathologist review. In addition, we have demonstrated that it is feasible to use VR-SIM to routinely image very large gross pathology specimens, such as entire prostate resection surfaces, in relatively short timeframes at subcellular resolution. However, our prior work has focused on applications in prostate cancer; the utility in other organ sites has not been explored.
Here we extended our technology to varying size kidney, liver, and lung biopsies. We conducted a validation study of VR-SIM against histopathology on a variety of human tissues, including both small biopsies and large slices of tissue. We conducted a blinded study in which the study pathologist accurately identified the organs based on VR-SIM images alone. The results were then used to create a clinical atlas between VR-SIM and H and E images for the different tissues of interest. This clinical atlas will be used to aid in pathologist interpretation in future POC clinical applications of VR-SIM in kidney, liver, and lung. Such applications could include on-site identification of the presence of kidney glomeruli for to ensure successful downstream IHC analysis, or determination of the adequacy of lung cancer biopsies for genomic analysis.
Histological assessment of freshly removed tissue specimens requires accurate and fast analysis in clinical procedures such as diagnostic biopsy and surgical tumor resection. Current histological assessment methods are either time-consuming or damage the tissue beyond the ability to re-analyze post-procedure. We demonstrate a novel dual-stain fluorescent analogue to brightfield Hematoxylin and Eosin for in-procedure histopathology that is both time-efficient and preserves the analyzed tissue for later analysis. H&E-like images are created from the combination of DRAQ5 and Eosin applied to human prostate tissue and animal muscle tissue under confocal microscopy. D&E images are pseduocolored to match H&E coloring, showing near-identical features to brightfield H&E of the same tissue. The histological accuracy, short staining time, and tissue preservation aspects of this dual-stain technique demonstrates its potential to be adopted for use in point-of-care pathology.
David Tulman, Mei Wang, Hillary Kimbrell, Andrew Sholl, Katherine Elfer, Tyler Schlichenmeyer, Sree Mandava, Benjamin Lee, Michelle Lacey, J. Quincy Brown
Intra-operative surgical margin assessment by pathology is labor-intensive and time-consuming and is not practically capable of sampling the entire specimen. Positive surgical margins (PSMs), or tumor extending to the surface of the excised specimen, are associated with increased tumor recurrence and are accepted as poor independent prognostic indicators. Considering the PSM rate is high for patients with prostate and kidney cancer, residual tumor following radical prostatectomy and partial nephrectomy remains a significant problem. To address the unmet clinical need for an imaging tool that can provide sub-cellular resolution images of large areas of excised surgical specimens in an intra-operative timeframe, we have developed a video rate structured illumination microscopy (VR-SIM) system. We conducted a clinical trial using VR-SIM to create gigapixel mosaics of entire margin surfaces for each specimen. In the ongoing study, 5 patients undergoing radical prostatectomy and 4 patients undergoing partial nephrectomy participated to have digital images of their surgical specimens reviewed in comparison to the pathology report. The surfaces of the intact, excised specimens were imaged in an appropriate timeframe and showed visualization of histopathologically relevant structures.
Video-rate structured illumination microscopy (VR-SIM) of fluorescently stained prostate biopsies is demonstrated as a potential tool for rapid diagnosis of prostate biopsies at the point of care. Images of entire biopsies at 1.3 micron lateral resolution are rendered in seconds, and pathologist review of the resulting images achieves 90% accuracy as compared to gold standard histopathology.
Reduction of warm ischemia time during partial nephrectomy (PN) is critical to minimizing ischemic damage and improving postoperative kidney function, while maintaining tumor resection efficacy. Recently, methods for localizing the effects of warm ischemia to the region of the tumor via selective clamping of higher-order segmental artery branches have been shown to have superior outcomes compared with clamping the main renal artery. However, artery identification can prolong operative time and increase the blood loss and reduce the positive effects of selective ischemia. Quantitative diffuse reflectance spectroscopy (DRS) can provide a convenient, real-time means to aid in artery identification during laparoscopic PN. The feasibility of quantitative DRS for real-time longitudinal measurement of tissue perfusion and vascular oxygenation in laparoscopic nephrectomy was investigated in vivo in six Yorkshire swine kidneys (n=three animals). DRS allowed for rapid identification of ischemic areas after selective vessel occlusion. In addition, the rates of ischemia induction and recovery were compared for main renal artery versus tertiary segmental artery occlusion, and it was found that the tertiary segmental artery occlusion trends toward faster recovery after ischemia, which suggests a potential benefit of selective ischemia. Quantitative DRS could provide a convenient and fast tool for artery identification and evaluation of the depth, spatial extent, and duration of selective tissue ischemia in laparoscopic PN.
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