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    Lymph node diagnostic excision


    Lymph node biopsies are commonly used in the investigation of lymphadenopathy for suspected lymphoma or tumours of unknown origin.1-3

    Intraoperative consultation is sometimes performed; imprints and/or smears are examined to determine the presence of tumour and may guide the extent of lymph node dissection required.

    This protocol includes lymph node diagnostic excisions. See separate protocols for core biopsies, sentinel and regional lymph node specimens from breast tumours and melanoma.

    Record the patient identifying information and any clinical information supplied together with the specimen description as designated on the container. See overview page for more detail on identification principles.

    • No
      • Non-routine fixation (not formalin), describe.
    • Yes
      • Special studies required, describe.
      • Ensure samples are taken prior to fixation.

    Fresh unfixed tissue should be handled in appropriate extraction cabinets and with suitable personal protection equipment for infection control. Unfixed specimens should be cut into 2mm slices perpendicular to the long axis, using a fresh, sterile blade for distribution to appropriate laboratories (internal and external). These may include frozen section, imprints, flow cytometry, cytogenetics, molecular laboratory, microbiology laboratory, tissue bank, electron microscopy and macroscopic photography.2

    Fresh lymph node sampling for flow cytometry

    Scraping the cut surface of a dissected lymph node with the cutting edge of sterile blade several times and placing the cell material into RPMI1640 (until the medium becomes cloudy) is a simple and effective technique for obtaining fresh lymph node tissue for flow cytometric evaluation and may improve diagnostic yield.4

    Lymph node specimens may be kept at room temperature for up to two hours. Specimens for flow cytometry can be maintained in sterile conditions at room temperature in Hanks* solution or RPMI# 1640 media for 2-24 hours. Fresh specimens for cytogenetics, particularly cell culture for metaphase fluorescence in situ hybridization (FISH) require rapid transportation (on the day of collection) in sterile RPMI 1640 media. Immunofluorescence transport media containing ammonium sulphate should not be used.2

    *Hanks solution is a balanced salt solution designed to maintain pH and osmotic balance.
    #RPMI is an abbreviation of Roswell Park Memorial Institute where the medium was developed. RPMI 1640 is a pH8 bicarbonate buffering system that supports cell culture particularly of lymphocytes.
    • Not performed
    • Performed, describe type and result
      • Frozen section
      • Imprints
      • Other, describe

    See general information for more detail on specimen handling procedures.

    Inspect the specimen and dictate a macroscopic description.

    External Inspection

    Describe the following features of the specimen:


    Record as stated by the clinician.

    Specimen site

    Record as described by the clinician.

    Specimen description

    • Collective size of tissue in three dimensions (mm)
    • Number of lymph nodes submitted
    • Size of each lymph node in three dimensions (mm)


    Section the lymph node(s) at 2mm intervals perpendicular to the long axis to promote fixation. To avoid distortion of the tissue, large lymph nodes (>15mm) may be cut into 4mm slices initially, then fixed for 30-60 mins before dissecting at 2 mm intervals.

    Internal Inspection

    Describe any cut surface abnormalities.


    Submit sections of all lymph nodes as follows:

    • Ideally the entire lymph node should be submitted to assess for areas of possible transformation in low grade lymphomas and improve diagnostic accuracy in cases where nodal disease may be focal (e.g. Hodgkin lymphoma).
    • Where the lymph node is large (>30mm) and only representative sections are taken, remaining tissue may be processed at the same time even if not examined microscopically immediately. This allows optimal material to be available for further testing. Prolonged fixation can reduce the antigenicity of tissues and result in unreliable immunohistochemistry and impair recovery of DNA from paraffin blocks.4
    • Representative sections may be submitted initially. However if lymphoma is suspected after microscopic examination, consideration should be given to submitting the remaining tissue.

    Where extracapsular extension is apparent or suspected, lines of dissection should extend through adjacent tissues to allow microscopic evaluation of extracapsular invasion.

    Record details of each cassette.

    An illustrated block key similar to the one provided may be useful.

    Block allocation key

    Cassette id Site No. of pieces
    A-E Lymph node serially sectioned  


    Associate Professors David Ellis and John Miliauskas for their contribution in reviewing and editing this protocol.


    1. Australian Cancer Network Diagnosis and Management of Lymphoma Guidelines Working Party. Guidelines for the Diagnosis and Management of Lymphoma. The Cancer Council Australia and Australian Cancer Network, Sydney, 2005.
    2. Norris D, Ellis D, Green M, Joske D, Macardle P, Miliauskas J, Spagnolo D and Turner J. Tumours of haematopoietic and lymphoid tissue structured reporting protocol, The Royal College of Pathologists of Australasia, Surry Hills, NSW, 2010.
    3. Ramsay A, Attygale A, Menon G, Naresh K and Wilkins B. Tissue pathways for lymph node, spleen and bone marrow trephine biopsy specimens, The Royal College of Pathologists, London, 2008.
    4. Miliauskas J. Lymph node sampling for flow cytometric analysis. Pathology - Journal of the RCPA. 2002;34(5):481.
    5. Ferrer I, Armstrong J, Capellari S, Parchi P, Arzberger T, Bell J, et al. Effects of formalin fixation, paraffin embedding, and time of storage on DNA preservation in brain tissue: a BrainNet Europe study. Brain Pathol 2007;17:297–303.

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      LN diag 1

      Fresh lymph node for special studies

      LN diag 2

      Enlarged lymph node serially sectioned

      LN diag 3

      Lymph node tissue for processing

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