Flow cytometry is increasingly used in small animal veterinary medicine to further characterize leukocytes in dogs and cats, also known as immunophenotyping. Flow cytometry is commonly used to determine cell origin (i.e., B versus T cells) in canine lymphoma, identify markers associated with lymphoma prognosis, distinguish reactive from neoplastic lymphocytosis, differentiate thymoma from mediastinal lymphoma, and support or rule out lymphoma when cytology is inconclusive. Sample cellularity and quality are critical for this test. This article covers sample requirements and tips for sample collection.<\/p>\n
Take-Home Points<\/strong><\/p>\n Flow cytometry is a diagnostic test that is increasingly being used in veterinary medicine to further characterize hematopoietic cells, including lymphocytes.1<\/sup> It is a laser-based analytic technique that measures multiple characteristics of cells based on their light-scatter and light-emitting properties, similar to laser-based in-house hematology analyzers.2<\/sup> However, in addition to measuring physical characteristics of cells such as size and internal complexity, as hematology analyzers do, flow cytometry measures fluorescence emitted from fluorochrome-labeled antibodies used in immunophenotyping leukocytes (TABLE<\/b><\/span><\/span>\u00a01<\/b><\/span>).1-3<\/sup><\/span><\/p>\n Flow cytometry is most often used to:<\/span><\/p>\n Flow cytometry is not a stand-alone test and should always be interpreted in combination with the clinical history, physical examination findings, morphologic findings (i.e., cytology), and other pertinent diagnostic information.1<\/sup><\/span><\/p>\n Knowing the immunophenotype is critical for prognosis in dogs with lymphoma and may also affect treatment decisions.2,3<\/sup> Technologies that can assign a certain lineage (i.e., B or T cell) to lymphocytes include immunohistochemistry (IHC), immunocytochemistry, flow cytometry, and polymerase chain reaction for antigen receptor rearrangement (PARR). In the past, IHC was considered the gold standard for immunophenotyping; however, it requires tissue biopsy. Flow cytometry has very high agreement (94%) with IHC for immunophenotyping of canine lymphoma.8<\/sup> It is also less invasive and less costly to the client, and in addition to immunophenotyping (i.e., B-cell versus T-cell lymphoma), it can identify markers (i.e., major histocompatibility complex [MHC] class II) or subtypes of lymphomas that affect prognosis (TABLE<\/b><\/span><\/span>\u00a02<\/b><\/span>).5-7<\/sup> As a result, flow cytometry has largely replaced IHC.<\/span><\/p>\n PARR is not recommended for lineage assignment <\/b>as a first-line test. PARR assesses lymphocyte antigen receptor gene rearrangement diversity (often referred to as lymphocyte clonality), and its use should be restricted to differentiating reactive from neoplastic lymphoid proliferations if microscopic evaluation (i.e., cytology, histopathology) and immunophenotyping using flow cytometry, immunocytochemistry, or IHC are inconclusive.8,9<\/sup><\/span><\/p>\n Differentiating a reactive lymphocytosis from one with a neoplastic cause can be difficult when small to intermediate lymphocytes in blood are only mildly to moderately increased (<15<\/span>\u2009<\/span>000 to 20<\/span>\u2009<\/span>000\/<\/span>\u00b5<\/span>L).10<\/sup> In both, lymphocytes appear mature and well differentiated on cytology, and neoplastic cells are often indistinguishable morphologically from \u201cnormal\u201d or reactive lymphocytes.<\/span><\/p>\n First, persistence (lasting more than 2 to 3 months) should be documented by ruling out transient physiologic or epinephrine-induced lymphocytosis, which usually occurs in young, very excited animals (more commonly cats) and usually resolves once the animal is allowed to calm down. Persistent reactive lymphocytosis is a common feature in dogs with chronic Ehrlichia canis <\/i>infection. Lymphocytes are often granulated lymphocytes and CD8+<\/sup> T cells. <\/i>Non-neoplastic lymphocytosis can also be seen, albeit less pronounced, in hypoadrenocorticism<\/a> (dogs), hyperthyroidism<\/a> (usually cats), and immune-mediated diseases (especially immune-mediated hemolytic anemia<\/a> in cats) as well as following vaccine administration. Lymphocytosis can also be paraneoplastic (e.g., thymoma).10<\/sup><\/span><\/p>\n Chronic lymphocytic leukemia (CLL) should be considered if lymphocytes in circulation have atypical morphology, if flow cytometric analysis demonstrates a phenotypically homogeneous expanded lymphocyte population (with or without aberrant antigen expression), or if a phenotype typically present in low numbers has increased (such as B-cell CLL in dogs).10,11<\/sup> Dogs should be screened for E canis<\/i> infection before performing supportive tests; E canis<\/i> infection can cause homogeneous expansion of CD8+<\/sup> T cells and can also rarely cause clonal expansion of lymphocytes.<\/span><\/p>\n Canine CLL is predominantly of T-cell origin; most are CD8+<\/sup> T cells, and many cases have large granulated lymphocyte morphology on cytology (fine magenta cytoplasmic granules).10-12<\/sup> Most feline CLLs are also of T-cell origin, with CD4+<\/sup> T-helper lymphocytes as the predominant subset.13<\/sup> B-cell CLL is more common in small-breed dogs, and approximately half of these affected dogs present with enlarged lymph nodes and\/or splenomegaly.14<\/sup> Most peripheral blood B-cell expansion in cats is polyclonal; therefore, B-cell lymphocytosis in a cat is less likely neoplastic in origin.15<\/sup> A polyclonal non-neoplastic or preneoplastic expansion of B cells has also been reported in English bulldogs.16<\/sup> Clinical presentation and flow cytometry findings can overlap between B-cell CLL and the English bulldog B-cell lymphocytosis syndrome, and additional testing for clonality via PARR is necessary for further distinction.14,16<\/sup> CLL has a slow onset and a more protracted clinical course that is usually stable over several months to even years.11-13,17,18<\/sup><\/span><\/p>\n Differentiating between leukemia and stage V lymphoma can be difficult and is usually based on clinical course, lack or presence of significant lymphadenopathy, degree of blood and bone marrow involvement, and immunophenotypic characteristics determined by flow cytometry (i.e., CD34 expression, presence or absence of lymphoid and myeloid markers, or MHC class II expression). It is important to note that neoplastic cells in acute leukemia (\u201cblasts\u201d) can be easily misinterpreted as being lymphoid in origin based on morphology alone. Therefore, flow cytometry is critical in lineage assignment as lymphoma and leukemia can present with either or both peripheral blood and nodal involvement; the prognostically important information is the type of malignancy present, rather than where it is found.<\/span><\/p>\n A detailed review of leukemia classification is beyond the scope of this article. In general, however, diagnosis and classification of canine acute leukemia require a combination of clinical and clinicopathologic data, cytomorphologic criteria, immunophenotyping by flow cytometry, and cytochemical staining.10,19<\/sup> It is noteworthy that 64% (16\/25) of dogs with acute myeloid leukemia demonstrated clonal and biclonal B- or T-cell receptor rearrangements in 1 study.20<\/sup> Therefore, PARR is currently not recommended to distinguish between acute leukemia of myeloid or lymphoid origin in dogs.<\/span><\/p>\n Flow cytometry is a noninvasive test to distinguish lymphoma from thymoma in dogs and cats with a mediastinal mass, usually in combination with clinical findings, diagnostic imaging, and cytology. Given that these 2 diseases are treated completely differently (i.e., systemic chemotherapy for lymphoma versus surgical resection of thymoma), pretreatment distinction between these entities is critical. Typically, more than 10% of lymphocytes aspirated from a thymoma are small CD4+<\/sup>\/CD8+<\/sup> double-positive T cells, whereas that proportion is much lower in mediastinal lymphoma (<2%).21<\/sup> Thymoma can be associated with expansion of normal T cells in the peripheral blood. Circulating T\u00a0cells will most commonly be a combination of mature CD4+<\/sup> and CD8+<\/sup> T cells, and in some cases T\u00a0cells that do not express either antigen.22<\/sup><\/span><\/p>\n The characterization of cytologically \u201ctricky\u201d nodes can be clarified with flow cytometry. Such nodes include, for example, enlarged lymph nodes with a predominance of small lymphocytes or an expansion of intermediate lymphocytes for which distinction between a \u201cnormal\u201d lymph node and a hyperplastic lymph node from a small- or medium-sized lymphoma is not possible based on cytology. Other examples include some variants of canine T-cell lymphoma (aggressive and indolent) and small-cell B-cell lymphoma. Occasionally, lymphoma cells can be very fragile and difficult to prepare for cytologic diagnosis (i.e., a smear with too many ruptured cells with rare intact large lymphocytes concerning for lymphoma). In these cases, a lymph node aspirate for flow cytometry can support a lymphoma diagnosis.<\/span><\/p>\n Until sample preservation can be further optimized, clinical flow cytometry in veterinary medicine requires intact, viable cells. Therefore, a freshly collected sample <\/b>should be submitted or shipped to the laboratory on the same day or overnight for Monday through Friday delivery. Laboratories performing flow cytometry may offer overnight shipping labels. Friday collection is discouraged unless it is unavoidable.<\/span><\/p>\n Your veterinary nurses have the ability to be in charge of sample collection and preparation. Have them read more below:<\/p>\n How to Collect and Prepare Samples for the Laboratory<\/a><\/p><\/blockquote>\n\n
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<\/a><\/p>\nClinical Indications for Flow Cytometry<\/h2>\n
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<\/a><\/p>\nImmunophenotyping of Lymphoma and Identification of Prognostic Markers<\/h3>\n
Persistent Lymphocytosis: Differentiation Between Reactive and Neoplastic Causes<\/h3>\n
Differentiation Between Lymphoma and Leukemia<\/h3>\n
Distinction Between Mediastinal Lymphoma and Thymoma<\/h3>\n
Further Characterization of \u201cTricky\u201d Nodes<\/h3>\n
Sample Requirements and Collection<\/h2>\n
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