The fixed brains were embedded in paraffin, and subsequently cut in sections of 5 µm. After deparaffinization with xylene and rehydration through graded ethanol series, the slides were cooked for 20 min in citrate buffer for antigen retrieval. Slides were then stained overnight at 4°C with anti-amyloid-β antibody (1:1000; Abcam ab2539) followed by a 1 hr room temperature incubation with biotinylated secondary antibody (1:300; Dako E0431). Immunodetection was visualized using an Avidin-Biotin Complex kit (Vector Laboratories, UK), and sections were counterstained with haematoxylin before mounting. The slides were digitized with an automatic bright field microscope (Philips Ultra Fast Scanner, Philips, the Netherlands) and assessed by one examiner (LPM) for positivity for amyloid-β.
Ultrafast scanner
Manufactured by Philips
Sourced in Netherlands, United States
The UltraFast Scanner is a high-performance laboratory equipment designed for rapid data acquisition. It features advanced scanning technology capable of capturing images and data at exceptional speeds. The core function of the UltraFast Scanner is to provide efficient and accurate data collection for various scientific and research applications.
Automatically generated - may contain errors
Lab products found in correlation
3 3 diaminobenzidine (dab), by Vector Laboratories (2 mentions)
Intellisite pathology solution, by Philips (2 mentions)
Ab2539, by Abcam (1 mentions)
Avidin biotin complex kit, by Vector Laboratories (1 mentions)
E0431, by Agilent Technologies (1 mentions)
Nanozoomer slide scanner, by Hamamatsu Photonics (1 mentions)
Nanozoomer 2.0 ht, by Hamamatsu Photonics (1 mentions)
Dako liquid dab substrate chromogen system, by Agilent Technologies (1 mentions)
Envision flex, by Agilent Technologies (1 mentions)
Autostainer link 48, by Agilent Technologies (1 mentions)
Pannoramic 250, by 3DHISTECH (1 mentions)
Iscan ht, by Roche (1 mentions)
Nanozoomer, by Hamamatsu Photonics (1 mentions)
Aperio at2, by Leica (1 mentions)
Imagepro image analysis software, by Media Cybernetics (1 mentions)
Ab21176, by Abcam (1 mentions)
Biotinylated anti rabbit igg, by Vector Laboratories (1 mentions)
Abc kit, by Vector Laboratories (1 mentions)
Ab32072, by Abcam (1 mentions)
Envision detection system, by Agilent Technologies (1 mentions)
22 protocols using ultrafast scanner
1
Immunohistochemical Detection of Amyloid-β
2
Immunostaining and Quantification in Rodents
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Immunostained tissue sections were assessed by bright field light microscopy. All stained tissue sections were scanned using a Hamamatsu NanoZoomer slide scanner (NanoZoomer 2.0 HT, scanning software NDP-Scan Vers. 2.5, Hamamatsu Photonics France, Swiss Office, Solothurn, Switzerland). For image-analysis-based quantification cerebellar and striatal tissue sections from rats and dogs stained for Ki67 and calbindin D-28k were scanned using a Philips slide scanner (Philips Ultra Fast Scanner, version 1.6, Image Management System version 2.2, Philips Digital Pathology, Best, the Netherlands). A 40× objective was used for scanning, resulting in a pixel size of 0.25 µm in x and y directions. Image-analysis-based quantification was performed using Definiens Developer XD software (Developer XD 2.0, Definiens AG, Munich, Germany), and image analysis algorithms were developed for each respective immunostaining. The algorithms quantified stain-positive areas and computed unit-length labelling indices.
3
Immunohistochemical Analysis of CCL21 and CCR7
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Tumor sections were stained with anti-CCL21 (clone: HPA051210) (Sigma-Aldrich, Steinheim, Germany) and CCR7 (Abcam, Cambridge, UK) antibodies. Extensive validation data for anti-CCL21 antibody (HPA051210) using IHC on various TMAs and western blots are accessible at the Human Protein Atlas portal [23 (link)]. Sections were dewaxed, rehydrated and were subjected to citrate pH6.0 (CCL21) or Tris/HCl-EDTA pH9 (CCR7) antigen retrieval. Sections stained for CCL21 expression were incubated with 5 % nonfat dry milk for 30 min at room temperature and incubated with anti-CCL21 (1:600) in 5 % ELK overnight at 4 °C. Sections stained for CCR7 expression were incubated 1.5 % BSA with anti-CCR7 (1:2000) overnight at 4 °C. Afterward sections were incubated with Immunologic Poly-HRP-GAM/R/R IgG (Leica Biosystems, Eindhoven, The Netherlands) and Dako liquid DAB+ Substrate-Chromogen System (Dako, Heverlee, Belgium). Scanning of the slides was performed by Philips Ultra Fast Scanner (Philips Healthcare, Eindhoven, Netherlands). Tonsil tissues, both regular and decalcified FFPE processed, were used as a control. All slides were evaluated by at least two experienced persons of whom one was a reference pathologist (PCWH).
4
Automating PD-L1 Assessment in Cancer
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A total of 130 H and E and their corresponding PD-L1 IHC slides were scanned and digitized at a resolution of 0.25 μm/pixel (40× magnification) using a Philips Ultra Fast Scanner (Philips, Eindhoven, The Netherlands). The images were initially acquired in the iSyntax format and then converted to the tagged image format file (TIFF) format using Philips’ proprietary algorithm. Eighty-two of these WSIs were randomly chosen as an independent test cohort. To ensure a balanced test cohort (i.e., 41 PD-L1+ and 41 PD-L1−), these slides were equally sampled from tumor PD-L1+ and PD-L1− cases [Table 1 ]. The remaining 48 were used as a training cohort to train our deep learning architecture [Figure 1a and b ].
5
Immunohistochemical Analysis of Mantle Cell Lymphoma
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The standard panel of antibodies examined in patients with MCL covered CD20, CD3, BCL2, BCL6, MYC, CD10, MUM1, Ki67, cyclin D1, SOX11, TdT, CD5, CD38 and PAX5 (BSAP). The antibodies clones along with the manufacturer are named in Table 3 .
The H&E slides were scanned using UltraFast Scanner (Philips IntelliSite Solution, USA) with DigiPath™ Professional Production Software (Xerox, Norwalk, CT, USA) and representative areas of each case were selected and microtomed into 8 µm thick sections and mounted onto 1-mm-thick calcium fluoride (CaF2) windows (Crystran, UK).
The list of antibodies used in the immunohistochemical analysis.
| Antibody | Clone | Company |
|---|---|---|
| BCL2 | 124 | DAKO |
| BCL6 | PG-B6p | DAKO |
| BSAP (PAX-5) | DAK-Pax5 | DAKO |
| CD3 | Polyclonal rabbit | DAKO |
| CD5 | 4C7 | DAKO |
| CD10 | 56C6 | DAKO |
| CD20 | L26 | DAKO |
| CD38 | SP149 | Cell Marque |
| Cyclin D1 | EP12 | DAKO |
| MYC | Y69 | VENTANA |
| Ki-67 | MIB-1 | DAKO |
| MUM-1 | MUM1p | DAKO |
| SOX-11 | MRQ-58 | Cell Marque |
| TdT | EP266 | DAKO |
The H&E slides were scanned using UltraFast Scanner (Philips IntelliSite Solution, USA) with DigiPath™ Professional Production Software (Xerox, Norwalk, CT, USA) and representative areas of each case were selected and microtomed into 8 µm thick sections and mounted onto 1-mm-thick calcium fluoride (CaF2) windows (Crystran, UK).
6
Digitization Errors in Whole Slide Imaging
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Various examples of errors were accumulated that occurred during the digitization of glass slides. The aim of this study was not to report the incidence of such errors. Only errors in which the WSI (eSlide) differed from the macro image of the original glass slide were collected for this study. Examples were solicited from various pathology laboratories in the USA, Canada, Europe, and Asia. Each example submitted required a detailed explanation, if available images to document the error (e.g., screenshot), and any potential clinical impact that resulted. Examples of errors were attained from different types of scanners including an Aperio AT2 (Leica), Ultra-Fast Scanner (Philips), Pannoramic 250 (3DHistech), Nanozoomer (Hamamatsu), and iScan HT (Roche). Errors received were categorized into technical (scanner) and/or operator (manual) related causes and further evaluated for similarities and differences. Actual slide labels in some cases are displayed in order to illustrate the error that occurred; however, we believe that individual patients cannot be identified solely from these images.
7
Quantification of Lung Metastatic Burden
8
Protein Expression and Immunostaining
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Proteins were extracted using Bicine/CHAPS lysis buffer. Lysates were then separated via gel electrophoresis (Bio-Rad) and transferred to PVDF membranes using standard protocols. MYC and FLuc protein levels were detected using an anti-MYC (ab32072, Abcam)51 (link) or anti-FLuc (ab21176, Abcam)52 (link) antibody, respectively. The blot was imaged using a LI-COR scanner and analyzed by ImageJ.
Paraffin-embedded tumor sections were deparaffinized by successive incubation in xylene, 95% ethanol, 90% ethanol, and 70% ethanol, followed by PBS. Epitopes were unmasked by steaming in DAKO antigen retrieval solution for 45 min and then rinsed twice in PBS. The sections were blocked using DAKO blocking solution, and then immunostained overnight at 4 °C using primary antibodies (MYC, 1:150, Epitomics). Sections were then washed with PBS and incubated with biotinylated anti-rabbit IgG (1:300, Vector Labs) for 30 min at room temperature, then with the ABC kit (Vector Labs) for 30 min at room temperature. Sections were developed using 3,3′-diaminobenzidine (DAB, Vector Labs), counterstained with hematoxylin, and mounted with Permount. Images were obtained on a Philips Ultrafast Scanner.
Paraffin-embedded tumor sections were deparaffinized by successive incubation in xylene, 95% ethanol, 90% ethanol, and 70% ethanol, followed by PBS. Epitopes were unmasked by steaming in DAKO antigen retrieval solution for 45 min and then rinsed twice in PBS. The sections were blocked using DAKO blocking solution, and then immunostained overnight at 4 °C using primary antibodies (MYC, 1:150, Epitomics). Sections were then washed with PBS and incubated with biotinylated anti-rabbit IgG (1:300, Vector Labs) for 30 min at room temperature, then with the ABC kit (Vector Labs) for 30 min at room temperature. Sections were developed using 3,3′-diaminobenzidine (DAB, Vector Labs), counterstained with hematoxylin, and mounted with Permount. Images were obtained on a Philips Ultrafast Scanner.
9
HER2 Immunohistochemistry and Digital Image Analysis
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Glass slides were scanned using Philips UltraFast Scanner (Philips, the Netherlands) at ×40 magnification with a single-focus layer. The tissue on slides was detected automatically with focus points to obtain the optimal image. Whole slide images were stored in a centralized server located at The Ohio State University's campus. HER2 IHCs were evaluated using the HER2-CONNECT algorithm in the Visiopharm Integrator System (Visiopharm, Hørsholm, Denmark) and recorded as a value from 0 to 1[12 (link)] [Figure 1 ].
HER2 DIA scores were categorized into four categories with the following cutoff values: (1) 0: connectivity = 0; (2) 1+: 00.49. Similar to the HER2 scores used by pathologists, a HER2 DIA score 0 and 1 + were defined as negative, 2 + as equivocal, and 3 + as positive based on preliminary analysis and previously reported data.
HER2 DIA scores were categorized into four categories with the following cutoff values: (1) 0: connectivity = 0; (2) 1+: 0
10
Breast Cancer Immunohistochemistry Quantification
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Full‐face sections were prepared from the selected formalin‐fixed paraffin‐embedded tissue blocks. The IHC staining was performed automatically using the clinically validated DAKO Cytomation EnVision+ detection system according to standard protocols [20 (link)]. The Ki67‐stained slides were scanned using two high‐resolution scanners i.e. Philips Ultrafast Scanner and Pannoramic 3DHISTECH Scanner to scan about 1,376 and 705 cases, respectively. The scanned slides were then uploaded to our WASABI server (a customized version of HistomicsTK [21 (link)]), which is used for marking annotations and visualizing WSIs.
Annotations were marked at region level and cell level to develop ML models for region segmentation and cell detection and classification. Region annotations included tumor, ductal carcinoma in situ (DCIS), and normal regions while cell annotation included Ki67‐positive tumor, Ki67‐negative tumor, positive nontumor, and negative nontumor cells. In total, we collected annotations for around 600 mm2 of tumor area, 140 mm2 of normal area, 380 mm2 of DCIS area, and 9,433, 26,308, 2,424, 19,223 of the four cell types, respectively.
Annotations were marked at region level and cell level to develop ML models for region segmentation and cell detection and classification. Region annotations included tumor, ductal carcinoma in situ (DCIS), and normal regions while cell annotation included Ki67‐positive tumor, Ki67‐negative tumor, positive nontumor, and negative nontumor cells. In total, we collected annotations for around 600 mm2 of tumor area, 140 mm2 of normal area, 380 mm2 of DCIS area, and 9,433, 26,308, 2,424, 19,223 of the four cell types, respectively.
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