Automated Hematoxylin and Eosin Staining with Leica AutoStainer XL v1

PURPOSE: To stain formalin fixed paraffin tissue for microscopic evaluation. Hematoxylin will stain the nuclei of the tissue blue/purple and Eosin will stain cytoplasmic elements a spectrum of brilliant pink. QUALITY CONTROL: Fixation: 10% Neutral Buffered Formalin Technique: Paraffin sections at 5 microns Control: PCRL PROCEDURE: Station #Time in StationExact Time?Solution Name185:00NoXylene175:00NoXylene165:00NoXylene150:20No100% Alcohol140:20No100% Alcohol130:20No95% Alcohol120:20No70% AlcoholWash 11:20NoWater83:30YesHematoxylinWash 20:30YesWaterWash 30:30YesWater90:10YesAcid AlcoholWash 40:30YesWater100:10YesAmmonia WaterWash 50:30YesWater112:00Yes95%70:20YesEosin60:30Yes95%50:30Yes100%40:30Yes100%30:30NoXylene21:00NoXylene11:00NoXylene RESULTS: Nuclei……………………………………………..……………Blue/Purple Cytoplasmic Elements………………………………….Shades of brilliant pink For digital storage and possible pathology review slides are scanned on the Leica Aperio slide scanner. The file output is .svs and can be viewed by downloading freely available software from Leica.

Exact Tile-Based Segmentation Inference for Images Larger than GPU Memory

We address the problem of performing exact (tiling-error free) out-of-core semantic segmentation inference of arbitrarily large images using fully convolutional neural networks (FCN). FCN models have the property that once a model is trained, it can be applied on arbitrarily sized images, although it is still constrained by the available GPU memory. This work is motivated by overcoming the GPU memory size constraint without numerically impacting the fnal result. Our approach is to select a tile size that will ft into GPU memory with a halo border of half the network receptive feld. Next, stride across the image by that tile size without the halo. The input tile halos will overlap, while the output tiles join exactly at the seams. Such an approach enables inference to be performed on whole slide microscopy images, such as those generated by a slide scanner. The novelty of this work is in documenting the formulas for determining tile size and stride and then validating them on U-Net and FC-DenseNet architectures. In addition, we quantify the errors due to tiling confgurations which do not satisfy the constraints, and we explore the use of architecture effective receptive felds to estimate the tiling parameters.

Rocky road to digital diagnostics: implementation issues and exhilarating experiences

Since 2007, we have gradually been building up infrastructure for digital pathology, starting with a whole slide scanner park to build up a digital archive to streamline doing multidisciplinary meetings, student teaching and research, culminating in a full digital diagnostic workflow where we are currently integrating artificial intelligence algorithms. In this paper, we highlight the different steps in this process towards digital diagnostics, which was at times a rocky road with definitely issues in implementation, but eventually an exciting new way to practice pathology in a more modern and efficient way where patient safety has clearly gone up.

Zooming for cells: Tele-education of histopathology residents during the COVID-19 pandemic

The recent COVID-19 pandemic moved education into the virtual world. We surveyed the effectiveness of conducting virtual tutorials via Zoom with attached microscope for pathology residents. Eleven respondents from the Department of Anatomical Pathology, Singapore General Hospital completed the survey. Many noted that the clarity of the slide images was equal to that of the usual slide sessions (91%). The audio reception was acceptable (100%). We found that 93% were satisfied with using Zoom, with 18% showing high satisfaction. We see this technology as a good add-on to face-to-face teaching, as it allows participation of residents posted away from teaching hospitals, and that this method of instruction should be kept as we move past the pandemic and its restrictions. We also see this as an alternative to the use of scanned digital slides, as not all teaching centers have a digital slide scanner, in addition to the issues of viewing restriction and storage of digital slides that need to be considered.

Can a digital slide scanner and viewing technique assist the visual scoring for the cytokinesis-block micronucleus cytome assay?

The cytokinesis-block micronucleus cytome (CBMNcyt) assay is a comprehensive method to measure DNA damage, cytostasis and cytotoxicity caused by nutritional, radiation and chemical factors. A slide imaging technique has been identified as a new method to assist with the visual scoring of cells for the CBMNcyt assay. A NanoZoomer S60 Digital Pathology slide scanner was used to view WIL2-NS cells treated with hydrogen peroxide (H2O2) and measure CBMNcyt assay biomarkers using a high-definition desktop computer screen. The H2O2-treated WIL2-NS cells were also scored visually using a standard light microscope, and the two visual scoring methods were compared. Good agreement was found between the scoring methods for all DNA damage indices (micronuclei, nucleoplasmic bridges and nuclear buds) and nuclear division index with correlation R values ranging from 0.438 to 0.789, P < 0.05. Apoptotic and necrotic cell frequency was lower for the NanoZoomer scoring method, but necrotic frequency correlated well with the direct visual microscope method (R = 0.703, P < 0.0001). Considerable advantages of the NanoZoomer scoring method compared to direct visual microscopy includes reduced scoring time, improved ergonomics and a reduction in scorer fatigue. This study indicates that a digital slide scanning and viewing technique may assist with visual scoring for the CBMNcyt assay and provides similar results to conventional direct visual scoring.

DEVELOPMENT OF A MICROSCOPY SLIDE SYSTEM OF BASIC SUBJECTS FOR INCLUSION IN E -STUDIES FOR MEDICAL AND DENTISTRY STUDENTS

INTRODUCTION Medical and dentistry studies are time-consuming and require patience and perseverance. The study of basic medical subjects including Histology with Cell biology and Embryology require the possibility for regular study of tissues and self-evaluation which should not be limited in time, and should not be limited to classroom time. Furthermore, official slide-sets for the above-mentioned subjects often contain incomplete cell, tissue and organ slides which do not allow a full understanding of the teaching subjects. Thus, our aim was the development of a microscopy slide system for basic medical subjects to be included in e-studies which are accessible to medical students anywhere and anytime MATERIALS AND METHODS We planned to use 220 slides in Histology and 80 slides in Embryology. For the scanning process a Glissando Slide Scanner was used (Objective Imaging Ltd., UK). Slide images were then processed using Adobe Photoshop 7.0.1 Update and Paint.Net Software. A description of the slides was evaluated by three tutors, independent experts and IT specialists and all slides were transferred/incorporated into the Riga Stradiņš University e-study system. RESULTS The result was the development of 786 high quality digitalised slides with easily changeable magnification for two subjects: 372 new slides for use in Histology for the Medical faculty (MF) and 290 new slides for use in Histology for the Dentistry faculty (DF); 65 new slides for use in Embryology for the MF and 59 slides for the DF. Additionally, the quality of 230 Histology slides and 99 Embryology slides previously scanned using a press scanner were improved. Furthermore, descriptions of each slide were given in Latvian and English for easier orientation and to facilitate independent study. Altogether, slide descriptions were prepared for 16 2nd semester labs and 15 3rd semester labs in MF Histology, while the DF received 14 descriptions for 1st semester labs and 8 descriptions for 2nd semester labs in this subject. In Embryology, 6 lab slide descriptions for the MF were used and 5 lab slide descriptions for the DF were used. All slides were also marked with a special university watermark. The slide-set introduction contained a warning to students regarding copy/reproduction rights . In the end, the addition of slide study to e-studies was very highly evaluated by Latvian and foreign students in student feed-back questionnaires at the end of their studies in basic medical subjects. CONCLUSION The inclusion of Histology and Embryology slide-sets in e-studies allows an absolutely indispensable possibility for modern, independent study in medicine, saving the time of students and tutors outside the auditorium and creates the possibility of increasing the number of students in the classes. The digitalisation of high quality slides allows the development of many variations of basic teaching slides and expands teaching explanations in this way. The existing digitalised slide-sets and their descriptions can easily be changed if needed. However, the minus of such digitalised slide-sets in e-studies is that violations of university property copy rights by students cannot be excluded. ACKNOWLEDGEMENT The Foundation of Boris and Inara Teterevi is highly acknowledged for its financial support of the development of digitalised e-studies slide-set Programs in Histology and Embryology at Riga Stradiņš University.

A Low Cost Approach to Specimen Level Imaging of Natural History Microscope Slides using a DSLR System

Specimen level imaging of microscope slides often uses automated digital microscopy systems, however, these systems are not always suitable for non-standard slides, which are frequently found in natural history collections. Currently, for these types of slides imaging requires the use of less automated alternatives. This paper presents a low cost option for imaging non-standard slides, by using a DSLR camera with a 1:1 - 5:1 macro lens mounted to a macro focusing rail and a flashbox. There was no noticeable difference in the image resolution between this DSLR-based system and the 5x magnification provided using an automated slide scanner. The DSLR-StackShot system, while only partially automated, enables the required flexibility and manual control required when imaging slides of varying sizes, thicknesses, and preservation types, as well as slides that are damaged or in poor condition.

Detection of breast cancer lymph node metastases in frozen sections with a point-of-care low-cost microscope scanner

BackgroundDetection of lymph node metastases is essential in breast cancer diagnostics and staging, affecting treatment and prognosis. Intraoperative microscopy analysis of sentinel lymph node frozen sections is standard for detection of axillary metastases, but requires access to a pathologist for sample analysis. Remote analysis of digitized samples is an alternative solution, but is limited by the requirement for high-end slide scanning equipment.ObjectiveTo determine whether the image quality achievable with a low-cost, miniature digital microscope scanner is sufficient for detection of metastases in breast cancer lymph node frozen sections.MethodsLymph node frozen sections from 79 breast cancer patients were digitized using a prototype miniature microscope scanner and a high-end slide scanner. Images were independently reviewed by two pathologists and results compared between devices with conventional light microscopy analysis as ground truth.ResultsDetection of metastases in the images acquired with the miniature scanner yielded an overall sensitivity of 91 % and specificity of 99 % and showed strong agreement when compared to light microscopy (k= 0.91). Strong agreement was also observed when results were compared to results from the high-end slide scanner (k= 0.94). A majority of discrepant cases were micrometastases and sections of which no anticytokeratin staining was available.ConclusionAccuracy of detection of metastatic cells in breast cancer sentinel lymph node frozen sections by visual analysis of samples digitized using low-cost, point-of-care microscopy is comparable to analysis of digital samples scanned using a high-end, whole slide scanner. This technique could potentially provide a workflow for digital diagnostics in resource-limited settings, facilitate sample analysis at the point-of-care and reduce the need for trained experts on-site during surgical procedures.