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.
Current methods for breast tumor margin detection are invasive, time consuming, and typically result in a reoperative rate of over 25%. This marks a clear clinical need to develop improved tools to intraoperatively differentiate negative versus positive tumor margins. Here, we utilize photoacoustic tomography (PAT), ultrasound (US), and inverted Selective Plane Illumination Microscopy (iSPIM) to assess breast tumor margins in eight human breast biopsies. Our PAT/US system consists of a tunable Nd:YAG laser (NT 300, EKSPLA) coupled with a 40MHz central frequency US probe (Vevo2100, FUJIFILM Visual Sonics). This system allows for the delivery of 10Hz, 5ns pulses with fluence of 40mJ/cm2 to the tissue with PAT and US axial resolutions of 125μm and 40μm, respectively. For this study, we used a linear stepper motor to acquire volumetric PAT/US images of the breast biopsies using 1100nm light to identify bloodrich “tumor” regions and 1210nm light to identify lipid-rich “healthy” regions. iSPIM (Applied Scientific Instrumentation) is an advanced microscopy technique with lateral resolution of 1.5μm and axial resolution of 7μm. We used 488nm laser excitation and acridine orange as a general comprehensive histology stain. Our results show that PAT/US can be used to identify lipid-rich regions, dense areas of arterioles and arteries, and other internal structures such as ducts. iSPIM images correlate well with histopathology slides and can verify nuclear features, cell type and density, stromal features, and microcalcifications. Together, this multimodality approach has the potential to improve tumor margin detection with a high degree of sensitivity and specificity.
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.
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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