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Appendix I: Biopsy Procedures

Updated 10 March 2009

Technical Suggestions for Biopsies of Lymph Node and related Hematopoietic Organs

The single greatest obstacle of accurate diagnosis and hence to appropriate treatment of lymphoid and related hematopoietic malignancies is unsuitable handling of specimens at the time of surgery. Ideally, the surgeon should consult the hospital pathologist prior to biopsy as input from both sources is necessary for the satisfactory handling of lymph nodes.

1. Lymph Node Biopsy

The surgeon should excise the largest node in a group of nodes whenever possible. The entire node should be removed intact with a minimal amount of crushing or handling. The specimen should be exposed to drying for the shortest time possible. If any time must elapse prior to processing the specimen in the pathology department, it should be wrapped in gauze soaked in normal saline and placed in the fridge at 4°C. During grossing in of the lymph node, a fresh cut surface can be used to prepare imprint preparations. These should be stained with the same procedure as that used for marrow aspirates. The preferred fixative is buffered formalin and the tissue should be fixed for appropriate lengths of time depending on the size of the tissue. B-Plus is a reasonable alternative fixative, but should only be used as a secondary fixative when sufficient tissue is available. B5 is not recommended for use due to its harmful effects on the environment. A representative portion of the lymph node should be sent as quickly as possible to the Immunology Laboratory of the BCCA, Vancouver Cancer Centre for flow cytometric analysis. The tissue should be sent at 4°C using wet ice or an ice pack in a sterile container with saline. Although a frozen section may be requested during surgery as a guide, definitive diagnosis of lymphoma by frozen section is virtually impossible. In addition, if the node is small, frozen section should be avoided at all cost, as it will interfere with the subsequent quality of the permanent sections and immunohistochemistry. If sufficient material is available, a portion of the lymph node should be snap-frozen in liquid nitrogen. This resource is invaluable should complex molecular genetic testing or gene expression profiling be required.

For all lymph nodes submitted fresh to the BCCA a cell suspension is prepared from the lymph node and processed for immunophenotypic analysis by flow cytometry, cytogenetics, gene rearrangement studies using fluorescence in situ hybridization (FISH), molecular genetic testing and other related studies. Importantly, cytogenetics and flow can only be performed using fresh material. The dedicated Lymphoma Pathology Program at the BCCA will routinely perform some or all of these tests as required to reach a definitive diagnosis. If the original slides, a copy of the pathology report and a representative paraffin block(s) are also submitted when it becomes available with a request for consultation, these results are then integrated into a final consultative report. This process is typically very expeditious. The Hematopathology Program at the BCCA has an extensive test menu for lymphoid malignancies, including many tests and reagents that are not commercially available. There is no charge for these and any related tests if cases are submitted for consultation.

2. Bone Marrow

Both a satisfactory aspirate and biopsy are required for the proper evaluation of patients with lymphoma. The aspirate is most useful in the diagnosis of leukemias and plasma cell disorders such as myeloma. Spread films, in addition to squash preparations, should always be made because these facilitate individual cell identification. A satisfactory trephine biopsy is essential for the diagnosis and staging of lymphoma as the location and shape of any lymphoid infiltrate is of major diagnostic importance.

3. Fine Needle Aspiration and Core Needle Biopsies

If at all possible an entire involved lymph node should be biopsied. This will provide a definitive diagnosis and proper subtyping which are essential for treatment planning. Fine needle aspiration biopsy or core needle biopsy may be quite useful in the re-staging and follow-up of patients with an established diagnosis of Hodgkin lymphoma or malignant lymphoma; however, a fine needle aspiration biopsy or needle core biopsy is not sufficient to establish the original diagnosis. If an involved lymph node cannot be completely excised, an incisional biopsy from involved tissue may be quite useful.

It is especially important not to place reliance on a negative fine needle aspiration biopsy or needle core biopsy in a patient with no established diagnosis. This often leads to delay in proper diagnosis.