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Pathology Core Laboratories (CHOP)

Director: Bhatti, Tricia R., MD

Summary:

The Pathology Core Laboratory at the Research Institute at Children's Hospital of Philadelphia unites several core pathology components in one facility. Path Core provides basic histopathology, research immunohistochemistry, tissue microarray, and laser capture microdissection services to researchers at Children's Hospital of Philadelphia and within the surrounding academic community. We offer a full range of histopathology services for both paraffin-embedded and frozen tissue samples including tissue processing, embedding, and cutting. We also perform most standard stains as well as immunohistochemistry, antibody workup, fluorescence, in situ hybridization and TUNEL. Tissue microarrays can be constructed and our staining services may be used on slides acquired from the arrays. Sophisticated imaging instrumentation is available for both bright field and fluorescent microscopy including whole slide scanning. We also host specialized software to analyze, manage, and store data on stained tissues and arrays.

Affiliations:

People:

Resources:

Databases

  • Antibody database ( Database )

    "This is a list of antibodies that we have worked up for IHC or IF staining for previous customers. We do not necessarily have everything on this list in stock. Although we do keep a handful of our more common markers in stock most of these were supplied by researchers for their project (who are sometimes willing to share). If you see something on the list that you are interested in let us know and we can help you find out if it would also work well for your project. If your marker is not on our list we can do a workup for your antibody on control tissue and help with validation before staining your experimental samples."

Instruments

  • Aperio ScanScope CS-O ( Slide scanner )

  • Aperio ScanScope IF ( Slide scanner )

  • Gallileo CK 3500 ( Tissue microarrayer )

    Computer driven tissue microarrayer

  • Leica Bond-Max autostainer ( Automatic staining machine )

  • Leica DM 4000B imaging microscope ( Microscope )

    "A Leica DM4000B upright scope paired with a Spot RT/SE Slider camera provides both brightfield and widefield fluorescence imaging capabilities. Optimized for 100micron coverslips.

    Objectives

    • 1.25x/N.A. 0.04
    • 5x/N.A. 0.15
    • 10x/N.A. 0.40
    • 20x/N.A. 0.70
    • 40x/N.A. 0.85
    • 63x/N.A. 0.90
    • 60x/N.A. 1.40-0.6 Oil (by appointment only)
    • 100x/N.A. 1.40-0.7 Oil (by appointment only)

    Filter cubes

    • Leica A4 – DAPI (Blue)
    • Leica L5 – FITC (Green)
    • Leica TX2- TxRd (Red)
    • Leica BGR – Triple Cube
    • Leica Y5 – Cy5 (Far Red)"

  • Leica DM2500 five-headed microscope ( Microscope )

  • Microm HM 325 ( Microtome )

  • Microm HM 335E ( Microtome )

  • Microm HM 355s ( Microtome )

    The core has two Microm HM 355s microtomes.

  • Microm HM 505E ( Cryostat )

  • Microm HM 560 ( Cryostat )

    "The Cryo-Star is an electronic, optionally motorized cryostat with retraction that incorporates a unique refrigeration system for independent specimen and knife cooling. This facilitates applications in both the histology and research labs.

    Other features include:
    • Automatic Cryo Approach (ACA) system for exact and safe approach
    of specimen towards the knife
    • Automatic cutting window (only with M and/or V version)
    • Ergonomic design of the unit allows user to work in either a standing
    or sitting position with padded and heated arm rests.
    • Intergrated vacutome system for stretching cryo-sections when
    cutting and for disposal of sections and debris when trimming (optional).
    • Electronic height adjustment (motorized stage only)"

  • Nikon inverted tissue culture microscope ( Inverted microscope )

  • Nikon Labophot-2 (2 headed) ( Microscope )

    Two headed optical microscope.

  • Sakura Cyto-Tek ( Cytospin centrifuge )

  • Sakura Tissue-Tek TEC ( Tissue embedding station )

  • Sakura Tissue-Tek VIP ( Automatic tissue processor )

    Vacuum infiltration tissue processor

  • ThermoFisher/Shandon Excelsior ( Automatic tissue processor )

  • ThermoFisher/Shandon Varistain Gemini ( Automatic staining machine )

  • Zeiss PALM Laser Capture Microdissection Microscope ( Laser capture microdissection microscope )

    The PALM microbeam system

    • Positioning Ablation Laser Micromanipulation (PALM)
    • Non-contact laser catapulting of tissue
    • Automation for efficient capture and pooling of large numbers of cells
    • Zeiss (Axiovert 200M) HotLInk Inverted scope offers a flexible platform for a variety of specimen types
    • Objective: 5x(non cutting), 10x, 20x, 40x, 63x (dry)
    • Fluorescence Filters: FITC, TxRd, Cy5

  • Zeiss stereomicroscope ( Stereo microscope )

Protocols

Services

  • Basic imaging workstation access ( Access service )

    "A Leica DM4000B upright scope paired with a Spot RT/SE Slider camera provides both brightfield and widefield fluorescence imaging capabilities. Optimized for 100micron coverslips."

  • Cut scrolls from block service ( Material processing service )

  • Decalcification service ( Material processing service )

  • Digital slide scanning service ( Material processing service )

    Digital slide scanning is available at 20x, 40x, and oil immersion magnifications.

    "Slide Scanning: Whole slides can be scanned and digitized to create "virtual slides" using the Aperio scanscope CS-O and IF scanners. The images are stored on a server making remote access possible. Slides can be viewed with any internet browser or else by using Image Scope."

  • Grossing service ( Material processing service )

  • H&E staining (on unstained slide) service ( Material processing service )

  • Histopathology technical consultation service ( Support service )

    Our staff is available for consultation, troubleshooting, and assistance in planning and interpreting histopathology experiments and related techniques.

  • Histopathology-related techniques training ( Training service )

    Training includes, but is not necessarily limited to, instruction for instrument use (how to use LCM), techniques (training a technician how to do immunostaining), and specialized topics (how to capture and analyze an image).

  • Immunohistochemistry services ( Material processing service )

    "The Core Lab can perform chromogenic or fluorescent labeling immunohistochemistry (IHC) by hand or using the latest in automated staining technologies. We use a Bond-Max auto-stainer from Leica Microsystems that allows us to run large batches of slides under uniform conditions."

  • In situ hybridization RNAscope service ( Material processing service )

    "RNAscope® is a novel multiplex nucleic acid in situ hybridization technology, based on ACD’s unique, patent-pending probe design and signal amplification methodology. It is the only clinically effective in situ RNA detection assay available today."

  • Laser capture microdissection microscope access ( Access service )

  • Laser capture service ( Material processing service )

    "The PALM system from Zeiss allows users to microdissect individual cells or regions of tissue from their slides for downstream molecular analysis.

    Why use laser dissection?
    Heterogeneous Nature of Tissue
    • Tumor Cells
    • Stroma
    • Lymphocytes

    Heterogeneous tissue may confound molecular analysis because it is impossible to determine which cells contribute to which cellular constituent of a given lysate. LMPC gives you the ability to isolate specific cells of interest without contamination from surrounding cells.

    Specimen requirements
    • OCT embedded frozen tissues
    • Paraffin embedded tissues
    • Cytology Preparations (i.e. blood smears, etc.)
    • Live Cell Cultures

    Isolation of DNA
    • Mutation analysis
    • Whole Genome Amplification
    • Chromosome Spreads
    • Select individual chromosomes for sequencing of translocation break points

    Isolation of RNA from cryosections
    • Quantitative RT-PCR
    • cDNA array hybridization
    • Chromosome Spreads

    Isolation of protein from specific cell populations
    • Amplification is not possible with protein samples so larger samples are needed
    • Highly sensitive methods of protein analysis (SELDI,MALD) are now available."

  • Microtome and embedding station access ( Access service )

    "The Pathology Core has seven microtomes and two embedding stations. One set is available for walk in customer use."

  • New antibody workup service ( Material processing service )

    "The core also provides an antibody "workup" services to test novel or untried antibodies on your tissue of interest. We have extensive experience testing antibodies in both human and animal tissues. Contact us to arrange a consultation."

  • Processing and embedding service ( Material processing service )

    "We accept wet fixed tissue specimens as well as frozen samples. Wet samples are processed on one of two automated tissue processors. The processing protocol is adjusted to match the size of the tissue specimen submitted. For instructions on submitting tissue samples please download our tissue submition guidelines."

  • Special stain (from slide) service ( Material processing service )

    • Level I
    • Level II
    • Level III
    • Special stain workup

  • Tissue microarray construction service ( Material processing service )

    "The pathology core can construct custom TMA's for researchers who have large collections of their tissue of interest and would like to be able to perform "high-throughput" analysis.

    Why do I need a TMA?

    • Basic research: Validate and verify gene expression data
    • Translational research: Diagnostic markers, prognostic markers, theraputic markers, response to therapy, query signaling pathways, drug discovery, tissue is a valuable resource

    What do I need to make a TMA?

    • Source tissue: animal tissue, clinical archival tissue, prospectively acquired clinical tissue, associated data
    • Pathologist: Confirm diagnosis, select representative areas for sampling (TMA cores are representative: 20 studies to date on a variety of tumors have shown strong correspondence in IHC results on TMAs when compared to "large" sections (n=3-4 cores), validate the constructed array, score the array.
    • Antibody or in situ probe to query array: phosphorylation state specific antibodies, signaling pathways
    • Data management and analysis tools
    • Willingness to have many collaborators

    TMA data analysis
    • IHC scoring
    — 0 negative
    — 1 indeterminate
    — 2 weakly positive
    — 3 strongly positive
    • Scores for each individual core in TMA are collected for every stain performed
    • Large data sets
    • Expression data can be analyzed by bioinformatic methods developed for cDNA microarrays
    • Quantitative scoring (pixel counts/percent area stained) can also be performed as well"

    Arrays that have been constructed previously can be found on the core's website.

  • TUNEL labeling service ( Material processing service )

  • Unstained slides preparation service ( Material processing service )

    Unstained slides can be prepared from paraffin blocks or frozen tissue.

    Complex sectioning is available (this is multiple non-sequential levels such as 3 sections per slide each section 50 microns apart).

  • Whole slide image analysis ( Data analysis service )

    "Whole slide image analysis is available using Aperio's "Image Analysis Toolbox" We can also perform analysis on site for IF images using the HALO platform from Indica Labs."

Software

  • Adobe Photoshop Lightroom ( Software )

    "Adobe Photoshop Lightroom software provides a comprehensive set of digital photography tools"

  • Aperature ( Software )

    "Aperature gives you everything you need to do so much more with your photos. Retouch and perfect images."

  • Final Cut Pro ( Software )

  • HALO ( Software )

    "We have a singe seat license (in lab use only) for HALO by Indica Labs. HALO is a whole slide image viewer and analysis platform. HALO adds additional features and tools for annotating images, making figures, and performing image analysis. Right now we are targeting this for users who would like to do whole slide analysis on slides scanned with our Aperio IF scanner. We have the following image analysis algorithms available so far: Area Quantification (brightfield and IF), Cytonuclear Quantification IF, and Microvessel Quantification IF."

  • Image-Pro Plus ( Software )

    "Image Pro provides image analysis tools such as cell counting and percent area stained measurements.'"

  • IP Lab ( Software )

  • Volocity ( Software )

    Volocity is a sophisiticated software for visualization, deconvolution, and quantitative analysis of 3- and 4-dimensional image data.


Web Links:

Last updated: 2016-06-21T14:53:16.552-04:00

Copyright © 2016 by the President and Fellows of Harvard College
The eagle-i Consortium is supported by NIH Grant #5U24RR029825-02 / Copyright 2016