Chapter 65 Although there are no published data systematically examining the incidence of lymphocytosis in a large population of dogs, analysis of 27,000 unique complete blood counts (CBCs) performed over a 4-year-period at the Veterinary Teaching Hospital at Colorado State University and over 100,000 CBCs performed through IDEXX Laboratories (Copland, n.d.) indicates that mild lymphocytosis occurs primarily in dogs that are 3 years old or younger. In the Colorado State University cases, only 0.6% of all CBCs performed showed lymphocytosis, and 45% of these cases were in dogs younger than 3 years. Although follow-up is not available presently for either of these two cohorts of patients, the data suggest that mild lymphocytosis in dogs is rare and is seen most commonly in younger patients. A flow cytometry study involves staining peripheral blood leukocytes with antibodies against a variety of cell surface proteins (Wilkerson, 2012). At a minimum, most laboratories determine whether the lymphocytes express T-cell antigens (CD3, CD4, CD5, and CD8), B-cell antigens (CD21 and CD22), and CD34, an antigen expressed on immature lymphoid and myeloid precursors. The flow cytometry results indicate the concentration of each lymphocyte subset in the peripheral blood and should be accompanied by an interpretation from the laboratory carrying out the assay. Many different laboratories now provide flow cytometry services, and most use a similar panel of antibodies. Flow cytometry is not a standardized assay in veterinary medicine, however, and there are no universally accepted protocols, normal ranges, or standard reporting format. For a clinician to get the most out of flow cytometry as a diagnostic tool, it is suggested that the clinician consistently use the same laboratory so that he or she can become familiar with that laboratory’s practices and reporting format. A third step in evaluating an expanded lymphocyte population may be determination of clonality using the PARR (polymerase chain reaction for antigen receptor rearrangements) assay. This assay is used when the flow cytometry study and cytologic analysis do not give definitive results. For example, flow cytometry might reveal an expansion of more than one lymphocyte subset, but a lymphoproliferative disorder still is strongly suspected based on clinical signs. Detection of clonality relies on the fact that lymphocytes contain DNA regions that are unique in length and sequence. This is a result of the recombination of the V, D, and J gene segments (B cell) or the V and J segments (T cell) to produce the region that encodes the antigen-binding portion of the immunoglobulin heavy chain or T-cell receptor-γ. Current methodology uses polymerase chain reaction to detect antigen receptor rearrangements, with primers for conserved regions of the V and J genes used to amplify the intervening region (Avery, 2012). Detection of clonal lymphocyte populations by this method is well established in human medicine and is routinely used in several veterinary laboratories. It is important to note that the conditions under which the assay is performed can have a profound effect on the sensitivity and specificity of the results; therefore each laboratory conducting the assay should report these numbers when they report results to allow proper interpretation of the findings. Lymphocytosis can occur in any type of lymphoproliferative disease, and traditionally there has been a distinction between leukemia (generally considered a disease of bone marrow origin that results in circulating lymphocytosis) and lymphoma (originating as a solid tumor). Recent developments in human medicine make this distinction less important. Rather, the contemporary human classification scheme describes more than 50 subtypes of leukemia and lymphoma, grouped instead by phenotype (B cell versus T cell) and by characteristic histologic, immunophenotypic, genetic, and epidemiologic features. Some of these diseases are classified as lymphoma, even though they commonly present with peripheral blood involvement, and others are classified as leukemia but may present primarily with nodal involvement. The clinically important diagnostic issue is determination of the subtype of neoplasia. For example, consider several B-cell lymphoproliferative disorders in people (Swerdlow, 2008). Mantle cell lymphoma is a B-cell tumor that presents with hepatic and splenic involvement, lymphadenopathy, and in most cases also lymphocytosis involving the neoplastic B cells. It is not thought to be curable. Diffuse large B-cell lymphoma also is a B-cell lymphoma that presents as a nodal or extranodal mass, but it only rarely includes lymphocytosis. The prognosis of this disease is different; it is associated with a longer median survival time and in some cases can be cured. These two diseases are distinguished by their characteristic histologic appearances, chromosomal abnormalities, and the constellation of cell surface antigens they express. They are both called lymphoma despite the fact that one of these diseases usually features blood involvement and the other one does not. As another example, chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma used to be considered two separate entities, one of which presents primarily as circulating lymphocytosis and one of which is primarily nodal. They are now considered a single disease that usually, but not always, features lymphocytosis and is characterized by mature B cells. The clinical course is indolent and can progress over many years, regardless of whether the patient has primarily nodal disease or blood disease at presentation. The only lymphoproliferative disorder than can be diagnosed definitively by immunophenotype is acute leukemia expressing CD34. CD34+ cells are precursor cells whose normal counterpart is found in the bone marrow. CD34 is expressed on both myeloid and lymphoid precursors. In most cases of CD34+ acute leukemia no antigens are expressed on the cell surface that would further allow for the definitive distinction between B, T, and myeloid lineages. If such a distinction is considered clinically relevant, the cells can be permeabilized and stained for intracellular expression of CD3 (for T-cell leukemia), Pax5 (for B-cell leukemia), or myeloperoxidase protein (for myeloid origin). The cells in these cases generally will be described by the cytopathologist as immature cells or blasts but occasionally can have a more mature appearance. Almost all cases of CD34+ leukemia show evidence of bone marrow involvement (anemia, thrombocytopenia, neutropenia) and can also show involvement of lymph nodes and spleen as well as a mediastinal mass at presentation. Regardless of the presentation and lineage designation, this form of acute leukemia has been shown to have a poor prognosis (Villiers et al, 2006; Williams et al, 2008).
Lymphocytosis in Dogs and Cats
Diagnosis
Diagnostic Testing
Flow Cytometry
Clonality Testing
Lymphoproliferative Disease in Dogs
Lymphocytosis Involving CD34+ Cells
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Lymphocytosis in Dogs and Cats
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