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What Happens to Your Tissue Specimen in Pathology?

It can be difficult for patients and other physicians to understand what happens to a specimen that the surgeon sends to pathology.One reason is that not all pathology laboratories at different hospitals process specimens the same way, and in the same time period. To clarify our process, we'll take you through the steps that will occur assuming that your biopsy is done here at Johns Hopkins Hospital. For simplicity, we will assume that it was done on a Monday morning.

  1. Accessioning (Monday morning)

    Accessioning Specimens As soon as your specimen arrives in pathology, your specimen is assigned a pathology number so we can keep track of it. This number is different from your medical record number or hospital identification number. The number begins with the letter S (for Surgical Pathology, as opposed to a C for cytology), followed by the last two numbers of the year. All of the specimens that arrive here in a given year are numbered consecutively starting from 1 (last year we received over 60,000 different specimens). If only one specimen is sent, it is assigned part 1. If the surgeon has sent other specimens from you separately (for example, the lumpectomy and the axillary lymph node dissection arrive in two different containers), the first specimen is designated part 1 while the second is assigned part 2.

    Therefore, your specimen might be assigned the number S01-12367, with part 1 being the lumpectomy and part 2 being an axillary dissection.

    If you have had specimens taken before at our hospital, they will have been given different pathology numbers. For example, a skin biopsy taken in 1984 might have the number S84-112. A pap smear (cytology) from this year might have the number C01-12854. A simple search of our computer system will let us know that you have had other specimens taken here before. This is important because we may need to review the slides from those specimens (which are kept permanently on file) when we evaluate the current specimen.

  2. Gross Examination (Monday morning)

    Gross Examination of Specimen Gross Examination is done within a few hours of receiving your specimen. A physician (pathologist 1, a resident) will examine your specimen, measuring its size, describing its color and texture, and determining if any masses are present. If so, the size of the mass and its distance to the specimens’ edge (the margin-where the surgeon cut) will be recorded.

    The pathologist then sections the specimen into thin slices with a scalpel and further examines the slices. The pathologist then takes small pieces of the slices (about the area of a dime and about twice as thick) and puts them into small containers called cassettes. Cassettes are essentially small, 2cmX2cmX0.5cm, rectangular boxes with multiple small openings in their walls which let the formalin fixative in to bathe the tissue. Once fixed and processed (see below), these pieces of tissue will be the blocks of the case.

    Open Cassette Closed Cassette
  3. Fixation (Monday afternoon)

    Multiple cassettes in a container of formalinThis is a crucial part of the process. In order to make microscopic slides, the tissue in the cassettes must be adequately treated in formalin, a fixative that hardens the tissue and prevents the proteins within the cells from degrading. Fixation allows us to save your tissue forever in our hospital (see below). The time needed for adequate fixation depends upon the size and fat content of the tissue, but it often can be completed in the afternoon.

    If the specimen comes late in the day, it may require overnight fixation, which will delay the diagnosis by one day. While we try to avoid delay, we feel that it is better to have a small delay than to proceed with inadequately fixed tissue. Inadequately fixed tissue can lead to:

    1. inability to make an adequate slide, particularly if the tissue is fatty. This make it impossible to assess margins, or determine how large the tumor is, both of which affect prognosis.
    2. unreliable immunohistochemical stains for prognostic markers like estrogen and progesterone receptor, Her2/neu, and Ki-67. If these proteins are not preserved properly by fixation, they degrade and become impossible to detect.

  4. Processing (Monday Evening)

    After the tissue is hardened by fixation, it is gradually and progressively dehydrated in alcohols overnight on a Tissue Processing Machine. These machines take about 8 hours to properly dehydrate the tissue.

  5. Embedding (Tuesday morning)

    Tissue BlocksThe processed tissue is then embedded in hot wax to form a tissue block. When the block hardens, it is stable at room temperature indefinitely.

    Tissue Block File Your tissue blocks are filed and saved forever in the pathology department at JHH, so at a later date a duplicate set of slides (recuts) can be made if you wish to have your pathology evaluated at another hospital. Also, if in the future you would like your tumor tested for a marker like Her2/neu, we can go back to the saved blocks and cut new slides for immunohistochemical staining. Also, if a new test or technological discovery comes along that would affect your breast cancer treatment in the future, and your doctor would like your tumor tested, we can go back to your tissue blocks and do the test.

  6. Making H and E Slides (Tuesday afternoon)

    A thin (5 micron) section of the tissue is cut from the block using a microtome (a cutting instrument), and stained with Hematoxylin and Eosin (H and E). The slides are now ready to be examined under the microscope, and are delivered to pathologist 1, who examined the fresh specimen.

    MicrotomeStaining MachineSlide
  7. Organization of Slides and Review by Pathologist 1 (Tuesday Evening)

    Pathologist 1 receives all of the slides corresponding to blocks (derived from cassettes) that he/she made the day before along with a typed dictation of his/her gross findings, and a code as to from where each section was taken. This pathologist reviews the slides, correlates the microscopic findings with the gross findings, and makes a provisional pathology report.

  8. Signout with Second Senior pathologist (Wednesday)

    The next day, pathologist 1 brings the provisional report and the slides to a second pathologist, one who is the attending pathologist for the case. The two pathologists review the slides together using a multi-headed microscope. The senior pathologist reviews and edits pathologist 1's diagnosis, and signs out (approves) the final diagnosis. If the findings are unusual, the senior pathologist may choose to review the case with other pathologists in the department at a daily quality assurance conference in order to be sure of the diagnosis.

    Once they have been reviewed by both pathologists and the case is signed out, the slides are kept in a file room at JHH indefinitely.

  9. Immunohistochemical stains

    Immunohistochemical stains These are stains done using antibodies to specific proteins in the cell. Proteins are composed of amino acids, and make up most of the structure of human cells. Antibodies are a specific type of protein which have the unique property of attaching to specific molecules. Antibodies are part of the normal human immune system, and function to protect us all from infection. Immunohistochemistry Laboratories take advantage of the unique attaching properties of antibodies. We use antibodies from mice or rabbits that specifically attach to human proteins like Estrogen receptor, Progesterone receptor, or Her2/neu, to detect these proteins in human cancer tissues.

    To do this test, the laboratory will cut several (about 5) additional sections from the block that the pathologist has designated. These slides will then be treated with bound labeled antibodies to the specific proteins we want to detect, treated with enzymes that detect the bound antibody by developing a brown colored chromagen, and then stained with hematoxylin. This will allow us to review the slide and see not only is the protein in question present but where it is (in the cancer or the normal benign cells).

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