Lymphocytosis in Dogs and Cats

Chapter 65


Lymphocytosis in Dogs and Cats





The term lymphocytosis refers to an absolute lymphocyte count above the reference range for the laboratory performing the count. For the majority of diagnostic laboratories, a lymphocyte count of more than 5000 cells/µl is considered above the reference range for dogs. It is clinically useful to distinguish isolated lymphocytosis, with minimal changes to other lineages, from mild and possibly artifactual lymphocytosis. The latter can be seen when there is marked leukocytosis secondary to severe inflammation or a significant response to other immunologic diseases such as hemolytic anemia. For example, practitioners should be cautious in overinterpreting a mild lymphocytosis in the context of a severe leukocytosis consisting primarily of neutrophils.


It also is important that clinicians not draw any conclusions from the presence of relative lymphocytosis when the total lymphocyte count is normal. When laboratories report differential counts, the results usually are accompanied by a normal range, and if the percentage of white cells that are lymphocytes is high, the result may be flagged as abnormal. However, there is no clinical significance to an increase in the percentage of lymphocytes in the blood when the total lymphocyte count is normal. Usually such an increase is the result of neutropenia, in which case the cause of the neutropenia should be addressed.


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.



Diagnosis



Differential Diagnoses


For mature dogs that have lymphocytosis but no evidence that other marrow elements are mobilized (i.e., no evidence of a strong erythroid regenerative response or marked neutrophilia) the list of differential diagnoses is short and can be investigated with readily available diagnostic tests. By far the most common reason for an adult dog to have isolated lymphocytosis is a lymphoproliferative disorder (lymphoma or leukemia). Additional but much less common differential diagnoses are thymoma, Addison’s disease, and chronic infection with Ehrlichia canis. There is little evidence in the literature that, in an adult dog, other infectious or inflammatory diseases cause lymphocytosis in the absence of significant anemia (e.g., lymphocytosis has been reported in cases of babesiosis, but these dogs also are anemic, and lymphocytes may be mobilized as a part of the bone marrow response to anemia).



Diagnostic Testing


Because a lymphoproliferative disorder is the most common reason for a mature adult dog to develop lymphocytosis, in the absence of clear signs pointing to a different cause (e.g., electrolyte abnormalities signaling Addison’s disease, or clinical signs suggesting a mediastinal mass), the most efficient diagnostic path begins with determining if the patient has lymphoma or leukemia. In canine patients a lymphocyte count higher than 20,000 cells/µl can be considered diagnostic for lymphoma or leukemia, and a presumptive diagnosis of neoplasia may be made on this observation alone. Further evaluation is warranted, however, because of the diverse outcomes and treatment differences associated with different categories of lymphoproliferative disorders.


The first step in evaluating a case of lymphocytosis, regardless of lymphocyte count, is cytologic review of the peripheral blood by a clinical pathologist. Such review is essential to verify lymphocytosis, especially if a differential count was obtained using automated equipment. Furthermore, the appearance of the cells provides essential information about the nature of the disease. Useful cytologic evaluations are descriptions of the size, uniformity, and nuclear and cytoplasmic characteristics, including cytoplasmic basophilia, granulation, chromatin texture, and the presence or absence of nucleoli. The presence of a predominant population of small mature lymphocytes is required for the diagnosis of chronic lymphocytic leukemia, whereas the presence of blasts and immature lymphocytes is more compatible with an acute leukemia.



Flow Cytometry


The second step in the evaluation of lymphocytosis is flow cytometry. Flow cytometry is performed on anticoagulated blood and provides information about the immunophenotype of the cells. The goal of a flow cytometry study is twofold: First, in cases in which the lymphocyte count is less than 20,000 cells/µl, flow cytometry can help determine if the lymphocyte population is homogeneous (i.e., consisting primarily of cells of a single phenotype), a finding that is most consistent with neoplasia. If the population is heterogeneous, other differential diagnoses listed earlier can be considered (the exception is E. canis infection, discussed later). Second, flow cytometry can determine the phenotype of the cells, which can help further classify the disease process and in some cases provide prognostic information.


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.



Clonality Testing


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.



Lymphoproliferative Disease in Dogs


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.


As suggested by these examples, a canine patient with lymphocytosis may have any number of lymphoproliferative disorders, most of which are not yet well described in the veterinary literature. The majority of these cannot be distinguished by examination of the peripheral blood alone, but cytologic examination and immunophenotyping of peripheral blood can give important prognostic and diagnostic information.



Lymphocytosis Involving CD34+ Cells


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).

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Jul 18, 2016 | Posted by in PHARMACOLOGY, TOXICOLOGY & THERAPEUTICS | Comments Off on Lymphocytosis in Dogs and Cats

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