Initial diagnosis of lymphoma involves determining whether the lymphocyte population in an affected organ appears normal and balanced in number, or abnormal and uniform in appearance indicating neoplasia (uncontrolled cell growth). The two types of lymphocytes involved in lymphoma are B lymphocytes, which manage humoral immunity, manufacturing antibodies that help the body recognize specific foreign invaders; and T lymphocytes, which manage cellular immunity, phagocytizing (ingesting) and killing foreign invaders.
Lymphoma often can be diagnosed with a fine needle aspirate. For this test, the veterinarian draws cells into a needle inserted into the suspicious lymph node or organ, and fixes the cells onto a slide for staining and microscopic examination. If the cells appear to be predominantly immature lymphocytes, we suspect lymphoma. Occasionally, verification of the diagnosis is performed by a pathologist who examines a lymph node biopsy (tissue sample).
Many patients start treatment following only these tests, but additional diagnostics are available to clarify and refine the diagnosis of lymphoma. These tests help differentiate reactive lymphocytosis (when the immune system is activated to fight infection) from neoplastic lymphocytosis (uncontrolled growth). In cases of neoplasia, they also identify the sub-type, or immunophenotype, of disease based on which type of lymphocyte (B cell versus T cell) is over-represented. Additional tests:
- Flow cytometry is run on live cells suspended in liquid (blood, cavity fluid samples, or aspirates suspended in saline). Antibodies attached to fluorescent dyes and specific for surface markers are applied to the sample cells. When labelled cells pass through a light source, the fluorescent intensity detected by the instrument correlates with the presence of that specific surface marker.
- Immunohistochemical staining is performed on cells from a fine needle aspirate or biopsy that have been fixed to a slide. Similar to flow cytometry, labelled antibodies bind to specific proteins on the cell surface, but they are visualized under the microscope instead of passed through a laser.
- PCR for antigen receptor rearrangements is an advanced test using a polymerase chain reaction technique to amplify DNA and assess the variable regions of B- and T-cell receptors. If it shows many cells with the same genes, they are suspected to be clones of a single cell, denoting malignancy.
Many factors determine which of these tests should be run for any individual patient. Flow cytometry and immunohistochemical staining provide more detail on the architecture of cells and therefore the sub-type of lymphoma present. Flow cytometry requires live cells, so samples must be chilled and sent overnight to the laboratory, but it has been shown to be more accurate than PCR for antigen receptor rearrangements when compared to immunohistochemical staining.2 PCR for antigen receptor rearrangements is prone to both false positive and false negative results, and has failed to positively identify an estimated 10 percent to 25 percent of lymphoma cases. However, it is the only test described here that can detect lymphoma when only a few neoplastic cells are present. If your dog is facing this diverse and sometimes confusing disease, ask your veterinarian to discuss with you all of the available diagnostic and treatment options, so you can work together and determine the best course of action for your dog.
- Avery, A. (2009). Molecular diagnostics of hematologic malignancies.Topics in companion animal medicine, 24(3), 144-150.
- Thalheim, L, Williams, LE, Borst, LB, Fogle, JE and Suter, SE. (2013). Lymphoma Immunophenotype of Dogs Determined by Immunohistochemistry, Flow Cytometry, and Polymerase Chain Reaction for Antigen Receptor Rearrangements. J Vet Intern Med, 27, 1509–1516.